Antibodies Against Amyloid Beta 4 With Glycosylated in the Variable Region

ABSTRACT

The present invention relates to a purified antibody molecule preparation being characterized in that at least one antigen binding site comprises a glycosylated asparagine (Asn) in the variable region of the heavy chain (V H ). More specifically, a pharmaceutical and a diagnostic composition comprising said antibody molecule and antibody mixtures are provided which is/are capable of specifically recognizing the β-A4 peptide/Aβ4. The present invention relates in particular to a mixture of antibodies comprising one or two glycosylated antigen binding sites with a glycosylated asparagine (Asn) in the variable region of the heavy chain, i.e. mixtures of isoforms of antibodies which comprise a glycosylated Asn in the variable region of the heavy chain (V H ). Also disclosed are compositions or antibody preparations comprising the specifically glycosylated antibody isoforms. Furthermore, the pharmaceutical and diagnostic uses for these antibodies are provided. The antibody isoforms may for example be used in the pharmaceutical intervention of amyloidogenesis or amyloid-plaque formation and/or in the diagnosis of the same.

The present invention relates to a purified antibody moleculepreparation being characterized in that at least one antigen bindingsite comprises a glycosylated asparagine (Asn) in the variable region ofthe heavy chain (V_(H)). More specifically, a purified antibody moleculeis provided which is capable of specifically recognizing the β-A4peptide/Aβ4 and comprising a glycosylation in the variable region of theheavy chain (V_(H)). The present invention relates to a mixture ofantibodies comprising one or two glycosylated antigen binding sites witha glycosylated asparagine (Asn) in the variable region of the heavychain, i.e. mixtures of isoforms of antibodies which comprise aglycosylated Asn in the variable region of the heavy chain (V_(H)). Alsodisclosed are compositions or antibody preparations comprising thespecifically glycosylated antibody isoforms. Furthermore, thepharmaceutical and diagnostic uses for these antibodies are provided.The antibody isoforms may for example be used in the pharmaceuticalintervention of amyloidogenesis or amyloid-plaque formation and/or inthe diagnosis of the same.

About 70% of all cases of dementia are due to Alzheimer's disease whichis associated with selective damage of brain regions and neural circuitscritical for cognition. Alzheimer's disease is characterized byneurofibrillary tangles in particular in pyramidal neurons of thehippocampus and numerous amyloid plaques containing mostly a dense coreof amyloid deposits and defused halos.

The extracellular neuritic plaques contain large amounts of apre-dominantly fibrillar peptide termed “amyloid β”, “A-beta”, “Aβ4”,“β-A4” or “Aβ”; see Selkoe (1994), Ann. Rev. Cell Biol. 10, 373-403, Koo(1999), PNAS Vol. 96, pp. 9989-9990, U.S. Pat. No. 4,666,829 or Glenner(1984), BBRC 12, 1131. This amyloid β is derived from “Alzheimerprecursor protein/β-amyloid precursor protein” (APP). APPs are integralmembrane glycoproteins (see Sisodia (1992), PNAS Vol. 89, pp. 6075) andare endoproteolytically cleaved within the Aβ sequence by a plasmamembrane protease, α-secretase (see Sisodia (1992), loc. cit.).Furthermore, further secretase activity, in particular β-secretase andγ-secretase activity leads to the extracellular release of amyloid-β(Aβ) comprising either 39 amino acids (Aβ39), 40 amino acids (Aβ40), 42amino acids (Aβ42) or 43 amino acids (Aβ43); see Sinha (1999), PNAS 96,11094-1053; Price (1998), Science 282, 1078 to 1083; WO 00/72880 orHardy (1997), TINS 20, 154.

It is of note that Aβ has several naturally occurring forms, whereby thehuman forms are referred to as the above mentioned Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43. The most prominent form, Aβ42, has the amino acidsequence (starting from the N-terminus):DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 3). In Aβ41,Aβ40, Aβ39, the C-terminal amino acids A, IA and VIA are missing,respectively. In the Aβ43-form an additional threonine residue iscomprised at the C-terminus of the above depicted sequence (SEQ ID NO:3).

The time required to nucleate Aβ40 fibrils was shown to be significantlylonger than that to nucleate Aβ42 fibrils; see Koo, loc. cit. and Harper(1997), Ann. Rev. Biochem. 66, 385-407. As reviewed in Wagner (1999), J.Clin. Invest. 104, 1239-1332, the Aβ42 is more frequently foundassociated with neuritic plaques and is considered to be morefibrillogenic in vitro. It was also suggested that Aβ42 serves as a“seed” in the nucleation-dependent polymerization of orderednon-crystalline Aβ peptides; Jarrett (1993), Cell 93, 1055-1058.

Modified APP processing and/or the generation of extracellular plaquescontaining proteinaceous depositions are not only known from Alzheimer'spathology but also from subjects suffering from other neurologicaland/or neurodegenerative disorders. These disorders comprise, interalia, Down's syndrome, Hereditary cerebral hemorrhage with amyloidosisDutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis),Creutzfeld Jacob disease, HIV-related dementia and motor neuropathy.

Until now, only limited medical intervention schemes for amyloid-relateddiseases have been described. For example, cholinesterase inhibitorslike galantamine, rivastigmine or donepezil have been discussed as beingbeneficial in Alzheimer's patients with only mild to moderate disease.However, also adverse events have been reported due to cholinergicaction of these drugs. While these cholinergic-enhancing treatments doproduce some symptomatic benefit, therapeutic response is notsatisfactory for the majority of patients treated. It has been estimatedthat significant cognitive improvement occurs in only about 5% oftreated patients and there is little evidence that treatmentsignificantly alters the course of this progressive disease.Consequently, there remains a tremendous clinical need for moreeffective treatments and in particular those which may arrest or delayprogression of the disease.

Also NMDA-receptor antagonists, like memantine, have been employed morerecently. However, adverse events have been reported due to thepharmacological activity. Further, such a treatment with theseNMDA-receptor antagonists can merely be considered as a symptomaticapproach and not a disease-modifying one

Also immunomodulation approaches for the treatment of amyloid-relateddisorders have been proposed. WO 99/27944 discloses conjugates thatcomprise parts of the Aβ peptide and carrier molecules whereby saidcarrier molecule should enhance an immune response. Another activeimmunization approach is mentioned in WO 00/72880, wherein also Aβfragments are employed to induce an immune response.

Also passive immunization approaches with general anti-Aβ antibodieshave been proposed in WO 99/27944 or WO 01/62801 and specific humanizedantibodies directed against portions of Aβ have been described in WO02/46237, WO 02/088306 and WO 02/088307. WO 00/77178 describesantibodies binding a transition state adopted by β-amyloid duringhydrolysis. WO 03/070760 discloses antibody molecules that recognize twodiscontinuous amino acid sequences on the Aβ peptide.

WO 03/016466 describes a humanized anti-Aβ antibody which is modified inorder to avoid any potential glycosylation in its heavy chain, since aglycosylation in variable region(s) of antibodies has been postulated inWallick (1988) J. Exp. Med. 168, 1099-1109.

The technical problem underlying the present invention is to provideefficacious means and methods in the medical management of amyloiddisorders, in particular means and methods for the reduction ofdetrimental amyloid plaques in patients in need of a (corresponding)medical intervention.

In a first aspect, the present invention provides a purified antibodymolecule being characterized in that at least one antigen binding sitecomprises a glycosylated asparagine (Asn) in the variable region of theheavy chain (V_(H)). The inventive, purified antibody or the antibodycomposition as provided herein is in particular directed against Aβand/or a fragment of Aβ. The purified antibody molecule as providedherein and in particular the antibody composition or antibodypreparation of the invention is useful in the preparation of apharmaceutical or diagnostic composition for the treatment, ameliorationand for prevention of a disease associated with amyloidosis and/oramyloid plaque formation. An example of such disease is Alzheimer'sdisease.

In context of this invention it was surprisingly found that purifiedantibody molecules, wherein at least one antigen binding site comprisesan N-linked glycosylation in the variable region of the heavy chain, isparticularly useful in e.g. the reduction of amyloid plaques.Furthermore, it has been found in context of this invention that theglycosylated antibodies or antibody compositions as provided herein areparticularly useful and efficacious in crossing the blood-brainbarrier/blood brain border in vivo as illustrated by very efficaciousplaque binding.

This is in stark contrast to the teachings of the prior art. WO03/016466 discloses antibodies that are specifically engineered to lackan N-glycosylation site in the heavy chain and it is taught thatglycosylation in variable region framework has negative effect onantibody binding affinity. It is taught in the prior art that thedescribed anti-Aβ antibody in a deglycosylated form of the heavy chainvariable CDR2 region has a markedly higher affinity for synthetic andpurified Aβ peptide in vitro.

Accordingly, the present invention relates to an improved, purifiedantibody molecule or an antibody preparation, in particular an antibodymolecule preparation that is directed against the Aβ4/Aβ peptide(amyloid β) and is highly efficient in vivo. The improvement of thepresent antibody molecule/antibody preparation lies in the provision ofpurified antibody molecules which comprise in at least one of theirvariable regions in the heavy chain an N-glycosylation, e.g. in the CDR2region of said variable region of the heavy chain. As mentioned above,this is in contrast to the prior art like WO 03/016466 that teaches thatsuch an N-glycosylation has to be avoided in antibodies directedagainst, e.g. Aβ.

Examples of an antibody molecule of the present invention areimmunoglobulin molecules, e.g. IgG molecules. IgGs are characterized incomprising two heavy and two light chains (illustrated for example inFIG. 14) and these molecules comprise two antigen binding sites. Saidantigen binding sites comprise “variable regions” consisting of parts ofthe heavy chains (V_(H)) and parts of the light chains (V_(L)). Theantigen-binding sites are formed by the juxtaposition of the V_(H) andV_(L) domains. For general information on antibody molecules orimmunoglobulin molecules see also common textbooks, like Abbas “Cellularand Molecular Immunology”, W.B. Sounders Company (2003).

In one aspect, for example in provision of an immunoglobulin molecule ascharacterized in this invention, an antibody is described wherein oneantigen binding site comprises a glycosylated asparagine (Asn) in thecorresponding variable region of the heavy chain (V_(H)). Said antibodyis hereinafter referred to as “mono-glycosylated ANTIBODY”; see alsoFIG. 14.

In another aspect, an immunoglobulin molecule is provided wherein bothantigen binding sites comprise a glycosylated asparagine (Asn) in thevariable region of corresponding heavy chains (V_(H)). Said antibodymolecule is hereinafter referred to as “double-glycosylated ANTIBODY”,see FIG. 14.

An immunoglobulin wherein no antigen binding site comprises aglycosylated asparagine (Asn) in the variable region of the heavy chain(V_(H)) is hereinafter referred to as “non-glycosylated ANTIBODY”.

The mono-glycosylated ANTIBODY, the double-glycosylated ANTIBODY and thenon-glycosylated ANTIBODY may comprise the identical amino acidsequences or different amino acid sequences. The term “ANTIBODY”comprises, accordingly, antibody molecules, in particular recombinantlyproduced antibody molecules, like immunoglobulins. However, as discussedbelow, the term “ANTIBODY molecule(s)” also comprises known isoforms andmodifications of immunoglobulins, like single-chain antibodies or singlechain Fv fragments (scAB/scFv) or bispecific antibody constructs, saidisoforms and modifications being characterized as comprising at leastone glycosylated V_(H) region as defined herein. A specific example ofsuch an isoform or modification may be a sc (single chain) antibody inthe format V_(H)-V_(L) or V_(L)-V_(H), wherein said V_(H) comprises theherein described glycosylation. Also bispecific scFvs are envisaged,e.g. in the format V_(H)-V_(L)-V_(H)-V_(L), V_(L)-V_(H)-V_(H)-V_(L),V_(H)-V_(L)-V_(L)-V_(H), which comprise the herein describedglycosylation in the CDR-2 region.

In context of this invention, the term “ANTIBODY” in capital letters isemployed in order to provide for better lucidity. However, the term“antibody” used in small letters is also used in context of thisapplication. “ANTIBODY”/“ANTIBODIES”/“antibody” and “antibodies” areused interchangeably.

The mono-glycosylated ANTIBODY and the double-glycosylated ANTIBODY areherein before referred to as “glycosylated ANTIBODY isoforms”. Apurified antibody molecule characterized in that at least one antigenbinding site comprises a glycosylated asparagine (Asn) in the variableregion of the heavy chain (V_(H)) is a mono-glycosylated ANTIBODY whichis free of or to a very low extent associated with an isoform selectedfrom a double-glycosylated ANTIBODY and a non-glycosylated ANTIBODY,i.e. a “purified mono-glycosylated ANTIBODY”. A double-glycosylatedANTIBODY in context of this invention is free of or to a very low extentassociated with an isoform selected from a mono-glycosylated ANTIBODYand a non-glycosylated ANTIBODY, i.e. a “purified double-glycosylatedANTIBODY”.

The term “which is free of or to a very low extent” denotes the completeabsence of the respective other (glycosylation) isoforms or a presenceof another (glycosylated) isoform in a concentration of at the most 10%,e.g. at the most 5%, e.g. at the most 4%, e.g. at the most 3%, e.g. atthe most 2%, e.g. at the most 1%, e.g. at the most 0.5%, e.g. at themost 0.3%, e.g. at the most 0.2%. Further information in this regard isprovided below and in the appended examples.

In context of this invention, the term “mono-glycosylated antibody(ies)”or “mono-glycosylated ANTIBODY(ES)” relates to antibody moleculescomprising an N-glycosylation in one (V_(H))-region of an individualantibody molecule, e.g. of an immunoglobulin, e.g. an IgG, e.g. of anIgG1. For example, said “mono-glycosylated form”, comprises aglycosylation on one variable region of the heavy chain e.g. at positionasparagine “Asn 52” as defined below. This “mono-glycosylated IgG1-formor mono-glycosylated isoform” may also comprise, as illustrated herein,the glycosylation in the well conserved glycosylation site in theFc-part, for example asparagine Asn 306 in the non-variable Fc-part.

The term “double-glycosylated antibody(ies)” or “double-glycosylatedANTIBODY(IES)” in the meaning of this invention comprises the hereindefined glycosylation on both variable regions of the heavy chain(V_(H))-region. Again, this “double glycosylated form”, comprises aglycosylation on the variable region of both heavy chains, in particularat position asparagine Asn 52 as detailed below and as exemplified inthe appended examples. This “double-glycosylated IgG1-form ordouble-glycosylated isoform” may also comprise, as illustrated herein,the glycosylation in the well conserved glycosylation site in thenon-variable/constant Fc-part, in particular on position 306 of theexemplified immunoglobulin. Appended FIG. 14 illustrates correspondingantibody molecules.

Antibodies devoid of such a post-translational modification in thevariable region, e.g. in both variable regions of the heavy chain (both(V_(H))-regions) are, in context of this invention considered as a“non-glycosylated form”, comprising no glycosylation in the variableregion of the heavy chain. Yet, this “non-glycosylated form” maynevertheless comprise (a) glycosylation(s) in the constant region(C-region) of the antibody, for example, and most commonly at the wellconserved glycosylation site of the Fc-part, in particular theasparagine (Asn) 306 in the non-variable/constant Fc-part as definedherein, see also SEQ ID NO: 6.

The glycosylated asparagine (Asn) in the variable region of the heavychain (V_(H)) may be in the complementarity determining region 2 (CDR2region). Said glycosylated asparagine (Asn) in the variable region ofthe heavy chain (V_(H)) may be in position 52 of the variable region asdefined below and as shown in SEQ ID No. 2 (or in position 52 of SEQ IDNO: 6 comprising also the Fc-part of an antibody heavy chain asdisclosed herein).

ANTIBODY isoforms may also comprise (a) further glycosylation(s) in theconstant/non-variable part of the antibody molecules, e.g. in theFc-part of an IgG, e.g. in the Fc-part in an IgG1. Said glycosylation inthe Fc-part relates to a well conserved glycosylation, beingcharacterized in located on position Asn306 of the heavy chain, e.g., inaccordance with the following sequence:

(SEQ ID NO: 6) QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

This sequence is also depicted herein below and the CDRs, CH-regions,heavy regions as well as two N-glycosylation sites (N52 and N306) areindicated:

The IgG-Fc region of the antibodies of this invention may be a homodimercomprised of inter-chain disulphide bonded hinge regions, glycosylatedC_(H)2 domains, bearing N-linked oligosaccharide at asparagine 306(Asn-306) of the C_(H)2 and non-covalently paired C_(H)3 domains. Theoligosaccharide of the glycosylation at Asn-306 is of the complexbiantennary type and may comprise a core heptasaccharide structure withvariable addition of outer arm sugars.

The oligosaccharide influences or determines Fc structure and function(Jefferis (1998) Immunol Rev. 163, 50-76). Effector functions, numberingparticular specific IgG-Fc/effector ligand interactions have beendiscussed (Jefferis (2002) Immunol Lett. 82(1-2), 57-65 and Krapp (2003)J Mol. Biol. 325(5), 979-89). This conserved Fc-position Asn-306corresponds to “Asn-297” in the Kabat-system (Kabat (1991) Sequences ofProteins of Immunological Interest, 5th Ed., Public Health Service,National Institutes of Health, Bethesda Md.)

The exemplified heavy chain may be encoded by the following sequence:

(SEQ ID NO: 5) caggtggaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcctccggatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagcgctattaatgcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaaacaccctgtatctgcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggtaatactcataagccttatggttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccagatatcgtgcgatatcgtgcaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga.

Said heavy chain may also comprise (in particular during its recombinantproduction) additional sequences, like “leader sequences”. Acorresponding example is encoded by the following sequence:

(SEQ ID NO: 25) atgaaacacctgtggttcttcctcctgctggtggcagctcccagatgggtcctgtcc (followed by) caggtggaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcctccggatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagcgctattaatgcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaaacaccctgtatctgcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggtaatactcataagccttatggttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgathe corresponding amino acid sequence would be:

(SEQ ID NO: 26) MKHLWFFLLLVAAPRWVLS (followed by) QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The above sequence also comprises a “signal peptide” said signal peptideis proteolytically cleaved by the host signal peptidase during thesecretory pathway during the production of the inventive antibodymolecules in host cells, like CHO cells.

Alternatively, said heavy chain may be encoded by a nucleic acidsequence that is optimized for recombinant production as exemplified bythe following sequence:

1 atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtga 51 ttcatggagaaatagagaga ctgagtgtga gtgaacatga gtgagaaaaa 101 ctggatttgt gtggcattttctgataacgg tgtccttctg tttgcaggtg 151 tccagtgtfollowed by

(SEQ ID NO: 23)         ca ggtggagctg gtggagtctg ggggaggcct ggtccagcct201 ggggggtccc tgagactctc ctgtgcagcg tctggattca ccttcagtag 251ctatgccatg agctgggtcc gccaggctcc aggcaagggg ctcgagtggg 301 tgtccgccataaacgccagc ggtacccgca cctactatgc agactccgtg 351 aagggccgat tcaccatctccagagacaat tccaagaaca cgctgtatct 401 gcaaatgaac agcctgagag ccgaggacacggctgtgtat tactgtgcga 451 gaggcaaggg gaacacccac aagccctacg gctacgtacgctactttgac 501 gtgtggggcc aaggaaccct ggtcaccgtc tcctcaggtg agtcctcaca551 acctctctcc tgcggccgca gcttgaagtc tgaggcagaa tcttgtccag 601ggtctatcgg actcttgtga gaattagggg ctgacagttg atggtgacaa 651 tttcagggtcagtgactgtc tggtttctct gaggtgagac tggaatatag 701 gtcaccttga agactaaagaggggtccagg ggcttttctg cacaggcagg 751 gaacagaatg tggaacaatg acttgaatggttgattcttg tgtgacacca 801 agaattggca taatgtctga gttgcccaag ggtgatcttagctagactct 851 ggggtttttg tcgggtacag aggaaaaacc cactattgtg attactatgc901 tatggactac tggggtcaag gaacctcagt caccgtctcc tcaggtaaga 951atggcctctc caggtcttta tttttaacct ttgttatgga gttttctgag 1001 cattgcagactaatcttgga tatttgccct gagggagccg gctgagagaa 1051 gttgggaaat aaatctgtctagggatctca gagcctttag gacagattat 1101 ctccacatct ttgaaaaact aagaatctgtgtgatggtgt tggtggagtc 1151 cctggatgat gggataggga ctttggaggc tcatttgagggagatgctaa 1201 aacaatccta tggctggagg gatagttggg gctgtagttg gagattttca1251 gtttttagaa tgaagtatta gctgcaatac ttcaaggacc acctctgtga 1301caaccatttt atacagtatc caggcatagg gacaaaaagt ggagtggggc 1351 actttctttagatttgtgag gaatgttcca cactagattg tttaaaactt 1401 catttgttgg aaggagctgtcttagtgatt gagtcaaggg agaaaggcat 1451 ctagcctcgg tctcaaaagg gtagttgctgtctagagagg tctggtggag 1501 cctgcaaaag tccagctttc aaaggaacac agaagtatgtgtatggaata 1551 ttagaagatg ttgcttttac tcttaagttg gttcctagga aaaatagtta1601 aatactgtga ctttaaaatg tgagagggtt ttcaagtact cattttttta 1651aatgtccaaa atttttgtca atcaatttga ggtcttgttt gtgtagaact 1701 gacattacttaaagtttaac cgaggaatgg gagtgaggct ctctcatacc 1751 ctattcagaa ctgacttttaacaataataa attaagttta aaatattttt 1801 aaatgaattg agcaatgttg agttgagtcaagatggccga tcagaaccgg 1851 aacacctgca gcagctggca ggaagcaggt catgtggcaaggctatttgg 1901 ggaagggaaa ataaaaccac taggtaaact tgtagctgtg gtttgaagaa1951 gtggttttga aacactctgt ccagccccac caaaccgaaa gtccaggctg 2001agcaaaacac cacctgggta atttgcattt ctaaaataag ttgaggattc 2051 agccgaaactggagaggtcc tcttttaact tattgagttc aaccttttaa 2101 ttttagcttg agtagttctagtttccccaa acttaagttt atcgacttct 2151 aaaatgtatt tagaattcga gctcggtacagctttctggg gcaggccagg 2201 cctgaccttg gctttggggc agggaggggg ctaaggtgaggcaggtggcg 2251 ccagcaggtg cacacccaat gcccatgagc ccagacactg gacgctgaac2301 ctcgcggaca gttaagaacc caggggcctc tgcgcctggg cccagctctg 2351tcccacaccg cggtcacatg gcaccacctc tcttgcagcc tccaccaagg 2401 gcccatcggtcttccccctg gcaccctcct ccaagagcac ctctgggggc 2451 acagcggccc tgggctgcctggtcaaggac tacttccccg aaccggtgac 2501 ggtgtcgtgg aactcaggcg ccctgaccagcggcgtgcac accttcccgg 2551 ctgtcctaca gtcctcagga ctctactccc tcagcagcgtggtgaccgtg 2601 ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa2651 gcccagcaac accaaggtgg acaagaaagt tggtgagagg ccagcacagg 2701gagggagggt gtctgctgga agccaggctc agcgctcctg cctggacgca 2751 tcccggctatgcagccccag tccagggcag caaggcaggc cccgtctgcc 2801 tcttcacccg gagcctctgcccgccccact catgctcagg gagagggtct 2851 tctggctttt tcccaggctc tgggcaggcacaggctaggt gcccctaacc 2901 caggccctgc acacaaaggg gcaggtgctg ggctcagacctgccaagagc 2951 catatccggg aggaccctgc ccctgaccta agcccacccc aaaggccaaa3001 ctctccactc cctcagctcg gacaccttct ctcctcccag attccagtaa 3051ctcccaatct tctctctgca gagcccaaat cttgtgacaa aactcacaca 3101 tgcccaccgtgcccaggtaa gccagcccag gcctcgccct ccagctcaag 3151 gcgggacagg tgccctagagtagcctgcat ccagggacag gccccagccg 3201 ggtgctgaca cgtccacctc catctcttcctcagcacctg aactcctggg 3251 gggaccgtca gtcttcctct tccccccaaa acccaaggacaccctcatga 3301 tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa3351 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 3401tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg 3451 tcagcgtcctcaccgtcctg caccaggact ggctgaatgg caaggagtac 3501 aagtgcaagg tctccaacaaagccctccca gcccccatcg agaaaaccat 3551 ctccaaagcc aaaggtggga cccgtggggtgcgagggcca catggacaga 3601 ggccggctcg gcccaccctc tgccctgaga gtgaccgctgtaccaacctc 3651 tgtccctaca gggcagcccc gagaaccaca ggtgtacacc ctgcccccat3701 cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 3751ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc 3801 ggagaacaactacaagacca cgcctcccgt gctggactcc gacggctcct 3851 tcttcctcta cagcaagctcaccgtggaca agagcaggtg gcagcagggg 3901 aacgtcttct catgctccgt gatgcatgaggctctgcaca accactacac 3951 gcagaagagc ctctccctgt ccccgggcaa atga

The “alternative” protein sequence as shown above as SEQ ID NO: 23comprises the same coding sequence as the first alternative, however ina slightly different genomic organization, like additional introns and aslightly different “leader sequence”/“signal sequence”. Said “leadersequence” may also comprise, as shown above an (additional) intron(s).The person skilled in the art is readily in a position to deduce in thesequence as shown herein the corresponding exon/intron structure byconventional methods.

The exemplified antibody described herein may also comprise a lightchain, said light-chain may comprise or have the following amino acidsequence

(SEQ ID NO: 8) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECwhich may be encoded by the following nucleic acid sequence:

(SEQ ID NO: 7) gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcgagccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcgcgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcagcctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttgaaattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag.

Also the “light chain” of exemplified antibody described herein maycomprise a “leader sequence” which is particularly useful in thetechnical production. A corresponding sequence may be (or may becomprised e.g. in a vector system) the following sequence:

(SEQ ID NO: 27) atggtgttgcagacccaggtcttcatttctctgttgctctggatctctggtgcctacggg (followed by) gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcgagccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcgcgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcagcctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttgaaattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

Said sequence encodes the following amino acid sequence

(SEQ ID NO: 28) MVLQTQVFISLLLWISGAYG (followed by) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Alternatively, said light chain may also be encoded by a nucleic acidsequence that is optimized for recombinant production as exemplified bythe following sequence:

1 atggacatga gggtcctcgc tcagctcctg gggctcctgc tgctctgttt 51 cccaggtaaggatggagaac actagcagtt tactcagccc agggtgctca 101 gtactgcttt actattcagggaaattctct tacaacatga ttaattgtgt 151 ggacatttgt ttttatgttt ccaatctcaggcgccagatg tfollowed by

(SEQ ID NO: 24)                                              gatatcgtg201 ttgacgcagt ctccagccac cctgtctttg tctccagggg aaagagccac 251cctctcctgc cgggccagtc agagtgttag cagcagctac ttagcctggt 301 accagcagaaacctggccag gcgcccaggc tcctcatcta tggcgcatcc 351 agcagggcca ctggcgtgccagccaggttc agtggcagtg ggtctgggac 401 agacttcact ctcaccatca gcagcctggagcctgaagat ttcgcgacct 451 attactgtct gcagatttac aacatgccta tcacgttcggccaagggacc 501 aaggtggaaa tcaaacgtga gtagaattta aactttgcgg ccgcctagac551 gtttaagtgg gagatttgga ggggatgagg aatgaaggaa cttcaggata 601gaaaagggct gaagtcaagt tcagctccta aaatggatgt gggagcaaac 651 tttgaagataaactgaatga cccagaggat gaaacagcgc agatcaaaga 701 ggggcctgga gctctgagaagagaaggaga ctcatccgtg ttgagtttcc 751 acaagtactg tcttgagttt tgcaataaaagtgggatagc agagttgagt 801 gagccgtagg ctgagttctc tcttttgtct cctaagtttttatgactaca 851 aaaatcagta gtatgtcctg aaataatcat taagctgttt gaaagtatga901 ctgcttgcca tgtagatacc atgtcttgct gaatgatcag aagaggtgtg 951actcttattc taaaatttgt cacaaaatgt caaaatgaga gactctgtag 1001 gaacgagtccttgacagaca gctcaagggg tttttttcct ttgtctcatt 1051 tctacatgaa agtaaatttgaaatgatctt ttttattata agagtagaaa 1101 tacagttggg tttgaactat atgttttaatggccacggtt ttgtaagaca 1151 tttggtcctt tgttttccca gttattactc gattgtaattttatatcgcc 1201 agcaatggac tgaaacggtc cgcaacctct tctttacaac tgggtgacct1251 cgcggctgtg ccagccattt ggcgttcacc ctgccgctaa gggccatgtg 1301aacccccgcg gtagcatccc ttgctccgcg tggaccactt tcctgaggca 1351 cagtgataggaacagagcca ctaatctgaa gagaacagag atgtgacaga 1401 ctacactaat gtgagaaaaacaaggaaagg gtgacttatt ggagatttca 1451 gaaataaaat gcatttatta ttatattcccttattttaat tttctattag 1501 ggaattagaa agggcataaa ctgctttatc cagtgttatattaaaagctt 1551 aatgtatata atcttttaga ggtaaaatct acagccagca aaagtcatgg1601 taaatattct ttgactgaac tctcactaaa ctcctctaaa ttatatgtca 1651tattaactgg ttaaattaat ataaatttgt gacatgacct taactggtta 1701 ggtaggatatttttcttcat gcaaaaatat gactaataat aatttagcac 1751 aaaaatattt cccaatactttaattctgtg atagaaaaat gtttaactca 1801 gctactataa tcccataatt ttgaaaactatttattagct tttgtgtttg 1851 acccttccct agccaaaggc aactatttaa ggaccctttaaaactcttga 1901 aactacttta gagtcattaa gttatttaac cacttttaat tactttaaaa1951 tgatgtcaat tcccttttaa ctattaattt attttaaggg gggaaaggct 2001gctcataatt ctattgtttt tcttggtaaa gaactctcag ttttcgtttt 2051 tactacctctgtcacccaag agttggcatc tcaacagagg ggactttccg 2101 agaggccatc tggcagttgcttaagatcag aagtgaagtc tgccagttcc 2151 tcccaggcag gtggcccaga ttacagttgacctgttctgg tgtggctaaa 2201 aattgtccca tgtggttaca aaccattaga ccagggtctgatgaattgct 2251 cagaatattt ctggacaccc aaatacagac cctggcttaa ggccctgtcc2301 atacagtagg tttagcttgg ctacaccaaa ggaagccata cagaggctaa 2351tatcagagta ttcttggaag agacaggaga aaatgaaagc cagtttctgc 2401 tcttaccttatgtgcttgtg ttcagactcc caaacatcag gagtgtcaga 2451 taaactggtc tgaatctctgtctgaagcat ggaactgaaa agaatgtagt 2501 ttcagggaag aaaggcaata gaaggaagcctgagaatacg gatcaattct 2551 aaactctgag ggggtcggat gacgtggcca ttctttgcctaaagcattga 2601 gtttactgca aggtcagaaa agcatgcaaa gccctcagaa tggctgcaaa2651 gagctccaac aaaacaattt agaactttat taaggaatag ggggaagcta 2701ggaagaaact caaaacatca agattttaaa tacgcttctt ggtctccttg 2751 ctataattatctgggataag catgctgttt tctgtctgtc cctaacatgc 2801 cctgtgatta tccgcaaacaacacacccaa gggcagaact ttgttactta 2851 aacaccatcc tgtttgcttc tttcctcaggaactgtggct gcaccatctg 2901 tcttcatctt cccgccatct gatgagcagt tgaaatctggaactgcctct 2951 gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg3001 gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag 3051agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 3101 agcaaagcagactacgagaa acacaaagtc tacgcctgcg aagtcaccca 3151 tcagggcctg agctcgcccgtcacaaagag cttcaacagg ggagagtgtt 3201 ag

The above “sequence” for an exemplified light chain has also a slightlydifferent genomic structure. This “alternative sequence” comprisesdifferent and/or additional introns. Accordingly the embodimentsdescribing for the “heavy chain” apply here mutatis mutandis.

In context of the present invention, the term “antibody molecule”relates to full immunoglobulin molecules, e.g. IgMs, IgDs, IgEs, IgAs orIgGs, like IgG1, IgG2, IgG2b, IgG3 or IgG4 as well as to parts of suchimmunoglobulin molecules, like Fab-fragments, Fab′-fragments,F(ab)₂-fragments, chimeric F(ab)₂ or chimeric Fab′ fragments, chimericFab-fragments or isolated V_(H)- or CDR-regions (said isolated V_(H)- orCDR-regions being, e.g. to be integrated or engineered in corresponding“framework(s)”) Also comprised in the term “antibody molecule” arediabodies and molecules that comprise an antibody Fc domain as a vehicleattached to at least one antigen binding moiety/peptide, e.g.peptibodies as described in WO 00/24782. Accordingly, and in context ofthis invention, the term “variable region of the heavy chain (V_(H))” isnot limited to a variable region in a full immunoglobulin but alsorelates to the corresponding parts of said variable region of the heavychain (V_(H)), like the CDRs, either alone or in combination of theCDR1, 2, and/or 3 or the corresponding “framework” of the variableregion. Therefore, an antibody molecule of the present invention mayalso be an antibody construct which comprises, as antigen binding site,the CDRs or at least one CDR of a given variable region of theglycosylated heavy chain (V_(H)). Said corresponding part of saidvariable region of the heavy chain (V_(H)) in the antibody construct ofthe invention is glycosylated as defined herein, e.g. comprises aglycosylated asparagine (Asn) in the antigen binding site. An example ofsuch an “isolated part” a variable region of the heavy chain (V_(H)) isthe herein exemplified CDR2 region comprised in SEQ ID NO: 12 (orencoded by a nucleic acid sequence as shown in SEQ ID NO: 11).

Furthermore, the term “antibody molecule” relates to modified and/oraltered antibody molecules, like chimeric, humanized or fully humanizedantibodies.

Said “fully humanized antibody” molecules are also characterized anddescribed as “completely human” antibodies. All these antibodies can begenerated by methods known in the art. For example, by phage displaytechnology, recombinant antibody molecules may be generated due to theuse of in vitro maturation which is the usage of a complete humanimmunoglobulin γ, subclass-1 framework (IgG1) as described by Knappik(2000) J Mol. Biol. 296(1), 57-86. and Rauchenberger (2003) J Biol.Chem. 278(40), 38194-205.

As documented in the appended examples, the term antibody, relates, e.g.to an IgG molecule and e.g. to an IgG1. The term also relates tomodified or altered monoclonal or polyclonal antibodies as well as torecombinantly or synthetically generated/synthesized antibodies. Theterm also relates to intact antibodies as well as to antibodyfragments/parts thereof, like, separated light and heavy chains, Fab,Fab/c, Fv, Fab′, F(ab′)₂. The term “antibody molecule” also comprisesantibody derivatives, the bifunctional antibodies and antibodyconstructs, like single chain Fvs (scFv) or antibody-fusion proteins.Also envisaged are catalytic and/or proteolytic antibodies whichcomprise a glycosylated V_(H) domain, e.g. a glycosylated V_(H)-CDR asdefined herein. The term “antibody molecule” relates also torecombinantly produced antibody molecules/antibody constructs which maycomprise, besides one specificity (e.g. against Aβ/Aβ), another or afurther specificity. Such constructs may comprise, but a not limited to“bi-specific” or “tri-specific” constructs. Further details on the term“antibody molecule” of the invention are provided herein below.

As pointed out above, also envisaged are a single-chain antibody, achimeric antibody, a CDR-grafted antibody, a bivalentantibody-construct, an antibody-fusion protein, a cross-cloned antibodyor a synthetic antibody comprising the herein defined glycosylation inat least one antigen binding site, e.g. in at least one variable regionof a/the heavy chain defined herein and being glycosylated. When forexample single-chain antibodies are produced, the herein defined“variable region of the heavy chain” is not limited to a heavy chain perse, but is meant to also relate to the corresponding parts derived froma heavy chain of a full antibody, e.g. a full immunoglobulin, like anIgG. Such parts may be the corresponding CDRs either alone or with partsof their corresponding framework. Furthermore, genetic variants ofimmunoglobulin genes are also envisaged in context of this invention.Genetic variants of, e.g., immunoglobulin heavy G chain subclass 1(IgG1) may comprise the Glm(17) or Glm(3) allotypic markers in the CH1domain, or the (Glm(1) or the Glm(non-1) allotypic marker in the CH3domain. Here, preferably an IgG1 of the Gm(17)(z) and Gm(1)(a) allotypeis employed. The antibody molecule of the invention also comprisesmodified or mutant antibodies, like mutant IgG with enhanced orattenuated Fc-receptor binding or complement activation. In oneembodiment, the antibody provided in accordance with this invention is afully-humanized antibody or “completely human” antibody.

Accordingly, the antibodies of the invention may also comprisecross-cloned antibodies, i.e. antibodies comprising different antibodyregions (e.g. CDR-regions) from one or more parental oraffinity-optimized antibody(ies) as described herein. These cross-clonedantibodies may be antibodies in several, different frameworks, e.g. anIgG-framework, e.g. a (human) IgG1-, IgG2a or an IgG2b-framework. Forexample, said antibody framework is a mammalian, e.g. a human framework.The domains on the light and heavy chains have the same generalstructure and each domain comprises four framework regions, whosesequences are relatively conserved, joined by three hypervariabledomains known as complementarity determining regions (CDR1-3).

As used herein, a “human framework region” relates to a framework regionthat is substantially identical (about 85% or more, usually 90-95% ormore) to the framework region of a naturally occurring humanimmunoglobulin. The framework region of an antibody (e.g. the combinedframework regions of the constituent light and heavy chains) serves toposition and align the CDR's. The CDR's are primarily responsible forbinding to an epitope of an antigen. It is of note that not onlycross-cloned antibodies described herein may be presented in a preferred(human) antibody framework, but also antibody molecules comprising CDRsfrom antibodies as described herein, may be introduced in animmunoglobulin framework. Examples for frameworks include IgG1, IgG2aand IgG2b. Most preferred are human frameworks and human IgG1frameworks, such as the heavy chain of an ANTIBODY as shown in, interalia, SEQ ID NO: 6.

In one embodiment ANTIBODY isoforms may comprise in the variable heavychain region a CDR1 comprising the following amino acids:

GFTFSSYAMS (SEQ ID NO: 10)

Said CDR1 may be encoded by the following nucleic acid sequence:

ggatttacctttagcagctatgcgatgagc (SEQ ID NO: 9)

ANTIBODY isoforms may comprise the following CDR2 in the variable regionof the heavy chain:

AINASGTRTYYADSVKG (SEQ ID NO: 12)(N: N-linked glycosylation site at Asn-52 of a Full Heavy Chain)

Said CDR2 may be encoded by the following nucleic acid sequence:

(SEQ ID NO: 11) gctattaatgcttctggtactcgtacttattatgctgattctgttaagg gt

The N-glycosylation in accordance with this invention is e.g. comprisedin said CDR2 region and is located on the corresponding Asn52 of thevariable region of the heavy chain, said variable region (V_(H)) beingencoded by a nucleic acid molecule as shown in SEQ ID NO: 1 and havingan amino acid sequence as shown in SEQ ID NO: 2.

Furthermore, ANTIBODY isoforms may comprise in their variable heavychain region a CDR3 comprising the following amino acid sequence:

GKGNTHKPYGYVRYFDV (SEQ ID NO: 14)

Said CDR3 may be encoded by the following nucleic acid sequence:

(SEQ ID NO: 13) ggtaagggtaatactcataagccttatggttatgttcgttattttgatgtt

ANTIBODY isoforms may comprise a light (L) chain which may becharacterized by the following CDRs:

CDR1: (SEQ ID NO: 16) RASQSVSSSYLA (SEQ ID NO: 15)agagcgagccagagcgtgagcagcagctatctggcg CDR2: (SEQ ID NO: 18) GASSRAT (SEQID NO: 17) ggcgcgagcagccgtgcaact CDR3: (SEQ ID NO: 20) LQIYNMPI (SEQ IDNO: 19) cttcagatttataatatgcctatt

ANTIBODY isoforms may comprise additional potential glycosylation sites(as known in the art comprising the Asn-X-Ser/Thr motives) in the aminoacid sequence of the heavy chains of the antibody, e.g. in the wellconserved glycosylation site at Asn 306 in the non-variable Fc-part(corresponding to “Asn297” in the Kabat-system (Kabat (1991) Sequencesof Proteins of Immunological Interest, 5th ed., Bethesda, Md.: NationalCenter for Biotechnology Information, National Library of Medicine),said heavy chains being or comprising the sequence as provided above,namely in SEQ ID NO: 6 (as encoded by SEQ ID NO: 5).

In one embodiment of the present invention, the ANTIBODY isoforms arecharacterized in that at least one antigen binding site comprises aglycosylated asparagine (Asn) in the variable region of the heavy chain(V_(H)), said V_(H) being encoded by

-   (a) a nucleic acid molecule comprising the nucleotide sequence as    shown in SEQ ID NO: 1:

(SEQ ID NO: 1) CAGGTGGAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATGCTTCTGGTACTCGTACTTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA;

-   (b) a nucleic acid molecule which encodes a polypeptide having the    amino acid sequence as shown in SEQ ID NO: 2:

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS (SEQ ID NO: 2; “N”in bold representing theherein defined Asn on position 52 of the variable region of the heavychain);

-   (c) a nucleic acid molecule that hybridizes to the nucleic acid    molecule of (a) or (b) and which encodes a polypeptide which is    capable of binding to the β-A4 peptide/Aβ4 as shown in the following    amino acid sequence

(SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

-    or a fragment thereof which comprises at least 15 amino acids;-   (d) a nucleic acid molecule that hybridizes to the nucleic acid    molecule of (a) or (b) and which encodes a polypeptide which is    capable of binding to at least two regions on the β-A4 peptide/Aβ4    as shown in the following amino acid sequence

(SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

-    or to at least two regions of a fragment of SEQ ID NO. 3 which    comprises at least 15 amino acids-    whereby said two regions on the β-A4 peptide Aβ4 or said fragment    thereof comprise the amino acids on position 3 to 6 and on position    18 to 26 of SEQ ID No. 3; or-   (e) a nucleic acid sequence that is degenerate to a nucleic acid    sequence as defined in any one of (a) to (d).

The person skilled in the art is aware of the fact that the term“nucleic acid molecule that hybridizes to the nucleic acid molecule of(a) or (b) and which encodes a polypeptide which is capable of bindingto at least two regions on the β-A4 peptide/Aβ4” as employed hereinrelates to a coding strand of a double stranded nucleic acid moleculewhereby the non-coding strand hybridizes to the above identified nucleicacid molecule of (a) and (b).

As pointed out above, the purified antibody molecule comprising theherein defined Asn-glycosylation may, inter alia, be characterized anddescribed as an antibody molecule wherein the variable region comprisinga glycosylated Asn is comprised in a heavy chain selected from the groupconsisting of:

-   -   (a) a heavy chain polypeptide encoded by a nucleic acid molecule        as shown in SEQ ID NOS: 5, 23 or 25;    -   (b) a heavy chain polypeptide having the amino acid sequence as        shown in SEQ ID NO: 6 or 26;    -   (c) a heavy chain polypeptide encoded by a nucleic acid molecule        that hybridizes to the nucleic acid molecule of (a) and which        encodes a polypeptide which is capable of binding to the β-A4        peptide/Aβ4 as shown in the following amino acid sequence

(SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

-   -    or a fragment thereof which comprises at least 15 amino acids;        or    -   (d) a heavy chain polypeptide encoded by a nucleic acid molecule        that hybridizes to the nucleic acid molecule of (a) and which        encodes a polypeptide which is capable of binding to at least        two regions on the β-A4 peptide/Aβ4 as shown in the following        amino acid sequence

(SEQ ID NO:3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

-   -    or to at least two regions of a fragment of SEQ ID NO. 3 which        comprises at least 15 amino acids        -   whereby said two regions on the β-A4 peptide Aβ4 or said            fragment thereof comprise the amino acids on position 3 to 6            and on position 18 to 26.

The above-identified antibody (e.g. an exemplified antibody of theinvention) may also comprise an L-chain with the following amino acidsequence:

(SEQ ID NO: 22) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECor an L-chain as, e.g. encoded by the following nucleic acid sequence:

(SEQ ID NO: 21) gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcgagccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcgcgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcagcctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttgaaattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

As printed out above, the purified antibody molecule comprising theherein defined Asn-glycosylation in the heavy chain may further comprisea light chain selected from the group consisting of:

-   -   (a) a light chain polypeptide encoded by a nucleic acid molecule        as shown in SEQ ID NOS: 7, 21, 24 or 27;    -   (b) a light chain polypeptide having the amino acid sequence as        shown in SEQ ID NO: 8, 22 or 28;    -   (c) a light chain polypeptide encoded by a nucleic acid molecule        that hybridizes to the nucleic acid molecule of (a) and which        encodes a polypeptide which is capable of binding to the β-A4        peptide/Aβ4 as shown in the following amino acid sequence

(SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFEAEDVGSNKGAIIGLMVGGVVIA

-   -    or a fragment thereof which comprises at least 15 amino acids;        or    -   (d) a light chain polypeptide encoded by a nucleic acid molecule        that hybridizes to the nucleic acid molecule of (a) and which        encodes a polypeptide which is capable of binding to at least        two regions on the β-A4 peptide/Aβ4 as shown in the following        amino acid sequence

(SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVLA

-   -    or to at least two regions of a fragment of SEQ ID NO. 3 which        comprises at least 15 amino acids

The term “hybridization” or “hybridizes” as used herein in context ofnucleic acid molecules/DNA sequences may relate to hybridizations understringent or non-stringent conditions. If not further specified, theconditions are preferably non-stringent. Said hybridization conditionsmay be established according to conventional protocols described, forexample, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”,Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, “Current Protocolsin Molecular Biology”, Green Publishing Associates and WileyInterscience, N.Y. (1989), or Higgins and Hames (Eds.) “Nucleic acidhybridization, a practical approach” IRL Press Oxford, Washington D.C.,(1985). The setting of conditions is well within the skill of theartisan and can be determined according to protocols described in theart. Thus, the detection of only specifically hybridizing sequences willusually require stringent hybridization and washing conditions such as0.1×SSC, 0.1% SDS at 65° C. Non-stringent hybridization conditions forthe detection of homologous or not exactly complementary sequences maybe set at 6×SSC, 1% SDS at 65° C. As is well known, the length of theprobe and the composition of the nucleic acid to be determinedconstitute further parameters of the hybridization conditions. Note thatvariations in the above conditions may be accomplished through theinclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility. Hybridizing nucleic acid molecules also comprisefragments of the above described molecules. Such fragments may representnucleic acid sequences which code for a non-functional antibody moleculeor a non-functional fragment thereof or for a CDR as defined herein, andwhich have a length of at least 12 nucleotides, preferably at least 15,more preferably at least 18, more preferably of at least 21 nucleotides,more preferably at least 30 nucleotides, even more preferably at least40 nucleotides and most preferably at least 60 nucleotides. Furthermore,nucleic acid molecules which hybridize with any of the aforementionednucleic acid molecules also include complementary fragments, derivativesand allelic variants of these molecules. Additionally, a hybridizationcomplex refers to a complex between two nucleic acid sequences by virtueof the formation of hydrogen bonds between complementary G and C basesand between complementary A and T bases; these hydrogen bonds may befurther stabilized by base stacking interactions. The two complementarynucleic acid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., Cot or Rotanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which, e.g., cellshave been fixed). The terms complementary or complementarity refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between single-stranded molecules. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term “hybridizing sequences” preferably refers to sequences whichdisplay a sequence identity of at least 40%, preferably at least 50%,more preferably at least 60%, even more preferably at least 70%,particularly preferred at least 80%, more particularly preferred atleast 90%, even more particularly preferred at least 95% and mostpreferably at least 97% identity with a nucleic acid sequence asdescribed above encoding an antibody molecule. Moreover, the term“hybridizing sequences” preferably refers to sequences encoding anantibody molecule having a sequence identity of at least 40%, preferablyat least 50%, more preferably at least 60%, even more preferably atleast 70%, particularly preferred at least 80%, more particularlypreferred at least 90%, even more particularly preferred at least 95%and most preferably at least 97% identity with an amino acid sequence ofthe antibody molecule as described herein above.

In accordance with the present invention, the term “identical” or“percent identity” in the context of two or more nucleic acid or aminoacid sequences, refers to two or more sequences or subsequences that arethe same, or that have a specified percentage of amino acid residues ornucleotides that are the same (e.g., 60% or 65% identity, preferably,70-95% identity, more preferably at least 95% identity), when comparedand aligned for maximum correspondence over a window of comparison, orover a designated region as measured using a sequence comparisonalgorithm as known in the art, or by manual alignment and visualinspection. Sequences having, for example, 60% to 95% or greatersequence identity are considered to be substantially identical. Such adefinition also applies to the complement of a test sequence. Preferablythe described identity exists over a region that is at least about 15 to25 amino acids or nucleotides in length, more preferably, over a regionthat is about 50 to 100 amino acids or nucleotides in length. Thosehaving skill in the art will know how to determine percent identitybetween/among sequences using, for example, algorithms such as thosebased on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994),4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), asknown in the art.

Although the FASTDB algorithm typically does not consider internalnon-matching deletions or additions in sequences, i.e., gaps, in itscalculation, this can be corrected manually to avoid an overestimationof the % identity. CLUSTALW, however, does take sequence gaps intoaccount in its identity calculations. Also available to those havingskill in this art are the BLAST and BLAST 2.0 algorithms (Altschul,Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36(1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410). TheBLASTN program for nucleic acid sequences uses as defaults a word length(W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoringmatrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) usesalignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

Moreover, the present invention also relates to nucleic acid moleculesthe sequence of which is degenerate in comparison with the sequence ofan above-described hybridizing molecule. When used in accordance withthe present invention the term “being degenerate as a result of thegenetic code” means that due to the redundancy of the genetic codedifferent nucleotide sequences code for the same amino acid.

In order to determine whether an amino acid residue or nucleotideresidue in a given antibody sequence corresponds to a certain positionin the amino acid sequence or nucleotide sequence of any of e.g. SEQ IDNOS: 1, 5, 23 and 25, the skilled person can use means and methodswell-known in the art, e.g., alignments, either manually or by usingcomputer programs such as those mentioned further down below inconnection with the definition of the term “hybridization” and degreesof homology.

For example, BLAST 2.0, which stands for Basic Local Alignment SearchTool BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.;Altschul (1990), loc. cit.), can be used to search for local sequencealignments. BLAST, as discussed above, produces alignments of bothnucleotide and amino acid sequences to determine sequence similarity.Because of the local nature of the alignments, BLAST is especiallyuseful in determining exact matches or in identifying similar sequences.The fundamental unit of BLAST algorithm output is the High-scoringSegment Pair (HSP). An HSP consists of two sequence fragments ofarbitrary but equal lengths whose alignment is locally maximal and forwhich the alignment score meets or exceeds a threshold or cutoff scoreset by the user. The BLAST approach is to look for HSPs between a querysequence and a database sequence, to evaluate the statisticalsignificance of any matches found, and to report only those matcheswhich satisfy the user-selected threshold of significance. The parameterE establishes the statistically significant threshold for reportingdatabase sequence matches. E is interpreted as the upper bound of theexpected frequency of chance occurrence of an HSP (or set of HSPs)within the context of the entire database search. Any database sequencewhose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.;Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used tosearch for identical or related molecules in nucleotide databases suchas GenBank or EMBL. This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score which is defined as:

$\frac{\% \mspace{14mu} {sequence}\mspace{14mu} {identity} \times \% \mspace{14mu} {maximum}\mspace{14mu} {BLAST}\mspace{14mu} {score}}{100}$

and it takes into account both the degree of similarity between twosequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Similar molecules are usually identified byselecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules. Another example for aprogram capable of generating sequence alignments is the CLUSTALWcomputer program (Thompson, Nucl. Acids Res. 2 (1994), 4673-4680) orFASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in theart.

In one embodiment the present invention provides glycosylated ANTIBODYisoforms wherein the glycosylation on Asn in the V_(H) region isselected from the group consisting of

-   (a) a sugar structure of the biantennary complex type without core    fucosylation;-   (b) a sugar structures of the biantennary hybrid type;-   (c) a sugar structures of the biantennary oligomannose type; and-   (d) a bi-antennary structure of any of the structures as provided in    appended FIG. 5 or appended FIG. 27.

The corresponding sugar structure does, in one embodiment of theantibody/antibodies of this invention not comprise a core fucosylation.

The corresponding N-glycosylation may predominantly consist of sugarstructures of the biantennary complex type (≧75%; mainly 80-90%) withoutcore fucosylation and highly sialidated with up to 80% of antennae.Minor sugar structures belong to the biantennary hybrid and theoligomannose type (≦25%), respectively and are also shown in appendedFIGS. 5 and 27. The glycosylation structures in the variable region areresistant to cleavage by N-glycosidase F from the protein (amino acidpolypeptide).

In one embodiment the dominant complex biantennary sugar structures arefurther characterized

-   -   by containing one or two sialic acids attached to either the one        or the other antenna or to both antennae. The sialic acid is of        the N-acetyl neuraminic acid type and is most likely bound in        alpha 2,3 linkage to the terminal beta 1,4 linked galactoses.    -   by lacking core fucosylation, i.e. lacking the fucose residue        attached in alpha 1,6 linkage to the innermost        N-acetyl-glucosamine at the reducing end of the sugar chain.

In one embodiment the hybrid sugar structures are further characterized

-   -   by containing a complex type antenna (a lactosaminyl unit        (GlcNAc-Gal) attached to the core sugar structure) as one arm of        the bi-antennary structure. This arm predominantly contains        N-acetyl neuraminic acid attached to the terminal beta 1,4        linked galactose.    -   by having one up to 3 additional mannose subunits attached to        the core sugar structure as the other antenna.    -   by lacking core fucosylation, i.e. lacking the fucose residue        attached in alpha 1,6 linkage to the innermost        N-acetyl-glucosamine at the reducing end of the sugar chain.

In one embodiment the oligomannose type sugar structures are furthercharacterized

-   -   by containing 4 (Man4→GlcNAc2), 5 (Man5→GlcNAc2) or 6        (Man6→GlcNAc2) mannose subunits in the complete sugar structure,        i.e. including the 3 branching mannose subunits present in a        typical N-linked core sugar structure.    -   by lacking core fucosylation, i.e. lacking the fucose residue        attached in alpha 1→6 linkage to the innermost        N-acetyl-glucosamine at the reducing end of the sugar chain.

In another embodiment of the present invention, a composition isprovided which comprises an antibody molecule being characterized inthat one antigen binding site comprises a glycosylated asparagine (Asn)in the variable region of the heavy chain (V_(H)) and an antibodymolecule being characterized in that two antigen binding sites comprisea glycosylated asparagine (Asn) in the variable region of the heavychain (V_(H)), i.e. a composition comprising mono-glycosylated ANTIBODYand double-glycosylated ANTIBODY, and is hereinafter referred to asANTIBODY COMPOSITION. The term ANTIBODY COMPOSITION also relates tocompositions which comprise molecules comprising at least oneglycosylated V_(H) region as defined herein or at least one glycosylatedCDR of said V_(H) region, whereby said molecules may, inter alia beimmunoglobulins or immunoglobulin isoforms and modifications asdescribed above. For example said composition may also comprise singlechain antibodies (scFvs) or bispecific molecules comprisingglycosylated, V_(H)-derived CDR regions.

Further definitions of the ANTIBODY COMPOSITION of this invention areprovided below.

The ANTIBODY COMPOSITION does not or does merely to a very low extentcomprise “in V_(H) non-glycosylated” antibody molecules, i.e. antibodiesthat do not comprise the herein defined glycosylation in the variableregion, in particular the variable part of the heavy chain (V_(H)).

In context of this invention and in particular in context of theantibody mixtures provided herein, the term “does not or does merely toa very low extent comprise non-glycosylated antibody molecules” meansthat the ANTIBODY COMPOSITION comprises less than 10%, e.g. less than5%, for example less than 4%, for example less than 3%, for example lessthan 2%, for example less than 1%, for example less than 0.5 or less ofthe/a non-glycosylated isoform as described herein.

Accordingly, in one embodiment, the present invention provides for anantibody preparation comprising mono-glycosylated and/ordouble-glycosylated antibodies (said glycosylation being located in thevariable region of the heavy chain) and being devoid of antibodymolecules without glycosylation in the variable region.

Again, the term “devoid of antibody molecules without glycosylation inthe variable region” relates to antibody preparations/antibodymixtures/antibody pools which comprise at the most 10%, e.g. at the most5%, e.g. at the most 4%, e.g. at the most 3%, e.g. at the most 2%, e.g.at the most 1%, e.g. at the most 0.5%, e.g. at the most 4%, e.g. at themost 3%, e.g. at the most 2%, e.g. at the most 1%, e.g. at the most0.5%, e.g. at the most 0.3%, e.g. at the most 0.2% non-glycosylatedisoforms as described herein.

In one embodiment the present invention provides a composition whichdoes not comprise more than 0.5% antibody isoforms which arenon-glycosylated in their variable regions, e.g. are non-glycosylated inthe variable region of the heavy chain.

As pointed out above, in one embodiment of the present invention, amixture of mono- and double-glycosylated antibodies, e.g.immunoglobulins, is provided, said mixture being devoid of antibodymolecules without glycosylation in the variable region. Antibodiesdevoid of such a post-translational modification in the variable region,e.g. in both variable regions of the heavy chain (both (V_(H))-regions)is, in context of this invention considered as an “non-glycosylatedform”, comprising no glycosylation in the variable region of the heavychain. Yet, this “non-glycosylated form” may nevertheless comprise (a)glycosylation(s) in the constant region (C-region) of the antibody, forexample, and most commonly at the well conserved glycosylation site ofthe Fc-part, in particular the asparagine (Asn) 306 in thenon-variable/constant Fc-part as defined herein.

The glycosylated ANTIBODY isoforms on their own or as a combination ofmono-glycosylated and double-glycosylated isoforms are very useful andadvantageous therapeutic antibody preparations for the treatment ofAlzheimer Disease (AD), and other amyloid related disorders like Down'ssyndrome, Hereditary cerebral hemorrhage with amyloidosis Dutch type,Parkinson's disease, ALS (amyotrophic lateral sclerosis), CreutzfeldJacob disease, HIV-related dementia and motor neuropathy. Theglycosylated ANTIBODY isoforms on their own or as a combination ofmono-glycosylated and double-glycosylated isoforms are also uniquediagnostic tools.

Both glycosylated isoforms as described herein show improved and highlyeffective brain penetration in vivo. Effective brain penetration andspecific binding to amyloid-β plaques can be demonstrated in PS2APPmice, a mouse model for AD-related amyloidosis.

Furthermore, improved specificity for genuine human amyloid-β plaques byimmunohistochemical stainings in vitro with significantly reducedunspecific stickiness could be detected. The minimal effectiveconcentration for consistent staining of human amyloid-β plaques wasdetermined to be 10 ng/ml, as documented in the appended examples.

As documented in the appended examples, the separation andcharacterization of differently glycosylated antibodies, e.g.immunoglobulins revealed that the glycosylation of the variable regionof the heavy chain has a surprising influence on the antigen binding toAβ peptides, the diagnostic value, the pharmacological profile andfunctional activity. The purified antibody molecules may be submitted toMS-analytics, binding studies (Biacore) and epitope mapping (Pepspotanalysis) binding to soluble Aβ, dissociation of aggregated Aβ andmicroscopical analysis of binding to β-amyloid plaques in vivo and invitro.

In one embodiment of the present invention, the purified ANTIBODY or theANTIBODY COMPOSITION is capable of specifically recognizing the β-A4peptide/Aβ4.

Accordingly, and as described herein, purified ANTIBODY or ANTIBODYCOMPOSITION relates in a specific embodiment to ANTIBODY or ANTIBODYCOMPOSITION capable of specifically recognizing two regions (theN-terminal region and the central/middle part) of Aβ/Aβ4.

The term “specifically recognizing” means in accordance with thisinvention that the antibody molecule is capable of specificallyinteracting with and/or binding to at least two amino acids of each ofthe two regions of β-A4 as defined herein. Said term relates to thespecificity of the antibody molecule, i.e. to its ability todiscriminate between the specific regions of the β-A4 peptide as definedherein and another, not related region of the β-A4 peptide or another,not APP-related protein/peptide/(unrelated) tests-peptide. Accordingly,specificity can be determined experimentally by methods known in the artand methods as disclosed and described herein. Such methods comprise,but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests andpeptide scans. Such methods also comprise the determination ofK_(D)-values as, inter alia, illustrated in the appended examples. Thepeptide scan (pepspot assay) is routinely employed to map linearepitopes in a polypeptide antigen. The primary sequence of thepolypeptide is synthesized successively on activated cellulose withpeptides overlapping one another. The recognition of certain peptides bythe antibody to be tested for its ability to detect or recognize aspecific antigen/epitope is scored by routine colour development(secondary antibody with an enzyme or a dye, like horseradish peroxidaseor 4-chloronaphthol or hydrogenperoxide), by a chemoluminescencereaction or similar means known in the art. In the case of, inter alia,chemoluminescence reactions or the use of a secondary fluorescentantibody, the reaction can be quantified. If the antibody reacts with acertain set of overlapping peptides one can deduce the minimum sequenceof amino acids that is necessary for reaction; see illustrative examplesprovided in accordance with this invention.

The same assay can reveal two distant clusters of reactive peptides,which indicate the recognition of a discontinuous, i.e. conformationalepitope in the antigenic polypeptide (Geysen (1986), Mol. Immunol. 23,709-715).

In addition to the pepspot assay, standard ELISA assay can be carriedout. As demonstrated in the appended examples small hexapeptides may becoupled to a protein and coated to an immunoplate and reacted withantibodies to be tested. The scoring may be carried out by standardcolour development (e.g. secondary antibody with horseradish peroxidaseand tetramethyl benzidine with hydrogenperoxide). The reaction incertain wells is scored by the optical density, for example at 450 nm.Typical background (=negative reaction) may be 0.1 OD, typical positivereaction may be 1 OD. This means the difference (ratio)positive/negative can be more than 10 fold. Further details are given inthe appended examples. Additional, quantitative methods for determiningthe specificity and the ability of “specifically recognizing” the hereindefined two regions of the β-A4 peptide are given herein below.

The term “two regions of the β-A4 peptide” relates to two regionsrelating e.g. to the N-terminal amino acids 3 to 6 and a central/middleepitope on position amino acids 18 to 24 of SEQ ID No. 3 (the β-A4peptide). As documented in the appended examples, in particular thedouble-glycosylated ANTIBODY A isoform provided and exemplified herein(see appended examples) detects two parts of the Aβ molecule, the firstpart comprising amino acid 1 to 10 in the N-terminus and the second partcomprising amino acids 17 to 26 of the central/middle part of Aβ (asshown in SEQ ID No. 3). Accordingly, in the antibody mixtures providedherein and comprising the mono- as well as the double-glycosylatedisoforms of the antibodies as provided herein, the two regions may alsosomewhat broadened, then comprising, e.g. amino acids 1 to 10 (or to 11or to 12) or a shorter part thereof and amino acids 17 to 26 (or aminoacids 16 to 27) or a shorter part comprised between amino acids 17 to26, like e.g. amino acids 19 to 26 or 20 to 26). The term “β-A4 peptide”in context of this invention relates to the herein above described Aβ39,Aβ41, Aβ43, in particular to Aβ40 and Aβ42. Aβ42 is also depicted inappended SEQ ID NO: 3. It is of note that the term “two regions of theβ-A4 peptide” also relates to an “epitope” and/or an “antigenicdeterminant” which comprises the herein defined two regions of the β-A4peptide or parts thereof. In accordance with this invention, said tworegions of the β-A4 peptide are separated (on the level of the aminoacid sequence) in the primary structure of the β-A4 peptide by at leastone amino acid, e.g. by at least two amino acids, e.g. by at least threeamino acids, e.g. by at least four amino acids, e.g. by at least fiveamino acids, e.g. by at least six amino acids. As shown herein and asdocumented in the appended examples, the inventive antibodies/antibodymolecules detect/interact with and/or bind to two regions of the β-A4peptide as defined herein, whereby said two regions are separated (onthe primary structure level of the amino acid sequence) by at least oneamino acid and wherein the sequence separating said tworegions/“epitope” may comprise more then seven, amino acids, more than 8amino acids, more than 10 amino acids or even about 14 amino acids.

The term “two regions of the β-A4 peptide” may also relate to aconformational epitope or a discontinuous epitope consisting of said tworegions or parts thereof; see also Geysen (1986), loc. cit. In contextof this invention, a conformational epitope is defined by two or morediscrete amino acid sequences separated in the primary sequence whichcome together on the surface when the polypeptide folds to the nativeprotein (Sela, (1969) Science 166, 1365 and Layer, (1990) Cell 61,553-6). The antibody molecules of the present invention are envisaged tospecifically bind to/interact with a conformational epitope(s) composedof and/or comprising the two regions of β-A4 described herein or partsthereof as disclosed herein below. The “antibody molecules” of thepresent invention are thought to comprise a simultaneous and independentdual specificity to (a) an amino acid stretch comprising amino acids 1to 11 (or (a) part(s) thereof) of β-A4 and (b) an amino acid stretchcomprising amino acids 16 to 27 (or (a) part(s) thereof) of β-A4 (SEQ IDNO. 3). Fragments or parts of these stretches comprise at least two, inmost cases at least three amino acids.

Antibody molecules, e.g. immunoglobulins could, inter alia, be expressedin three systems: a) in transiently transfected human embryonic kidneycells containing the Epstein barr virus nuclear antigen (HEK 293 EBNA,Invitrogen), b) in transiently transfected Chinese hamster ovary cells(CHO), and c) in stably transfected CHO cell lines (CHO K1 and CHO K1SV, Lonza Biologics). The three different antibody molecules (non-, monoor double-glycosylated) may be separated by specific purification steps,comprising protein A purification, cation exchange chromatography aswell as size column separation as detailed below.

In one embodiment of the invention, the antibody molecule isrecombinantly produced, e.g. in a CHO-cell or in a HEK 293 cell,preferably CHO-cells. In a particular embodiment the above identifiedglycosylation patterns may be obtained after expression in CHO-cells.CHO-cells are very well known in the art and comprise, inter alia, theCHO-cells as employed in the experimental part, like CHO K1 or CHO K1 SVcells. Commonly used HEK 293 cells are HEK 293 EBNA.

The recombinant expression of the glycosylated, inventive antibody iscarried out, as shown in the examples in a eukaryotic expression systemin particular in CHO-cells. However, further expression cells, i.e.eukaryotic cells may be envisaged. Eukaryotic cells comprise, forexample, fungal or animal cells. Examples for suitable fungal cells areyeast cells, e.g. those of the genus Saccharomyces, e.g. those of thespecies Saccharomyces cerevisiae. Suitable animal cells are, forinstance, insect cells, vertebrate cells, e.g. mammalian cells, such ase.g. NSO, MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR,NT2N, Sk-n-sh, CaSki, C33A. Also human cell lines are envisaged. Thesehost cells, e.g. CHO-cells, provide post-translational modifications tothe antibody molecules of the invention, including leader peptide orsignal sequence removal, folding and assembly of H (heavy) and L (light)chains and most importantly glycosylation of the molecule at correctsides, namely in the variable region of the heavy chain. Such signalpeptide or leader sequence is proteolytically cleaved by the host signalpeptidase during the secretory pathway during its recombinant productione.g. in CHO cells. Further suitable cell lines known in the art areobtainable from cell line depositories, like the American Type CultureCollection (ATCC). In accordance with the present invention, it isfurthermore envisaged that primary cells/cell cultures may function ashost cells. Said cells are in particular derived from insects (likeinsects of the species Drosophila or Blatta) or mammals (like human,swine, mouse or rat). Said host cells may also comprise cells fromand/or derived from cell lines like neuroblastoma cell lines.

Accordingly, the antibody molecule of the invention is prepared using arecombinant expression system. An example for such system, as pointedout above, is a mammalian expression system using Chinese hamster ovary(CHO) cells. These may be used with the glutamine synthetase (GS) system(WO 87/04462; WO 89/01036; Bebbington, 1992, Biotechnology (NY), 10,169-75). This system involves the transfection of a CHO cell with a geneencoding the GS enzyme and the desired antibody genes. CHO cells arethen selected which grow in glutamine free media and are also subjectedto inhibition of the GS enzyme using methionine sulphoximine (MSX). Inorder to survive, the cells will amplify the GS enzyme expression andconcomitantly the expression of the mAb.

Another possible expression system is the CHO dhfr− system, where theCHO cells are deficient for dihydrofolate reductase (dhfr−) anddependent on thymidine and hypoxanthine for growth. The parenteral CHOdhfr− cell line is transfected with the antibody and the dhfr gene thusenabling the selection of CHO cell transformants of the dhfr+ phenotype.Selection is carried out in the absence of thymidine and hypoxanthine.Expression of the antibody gene may be increased by amplification usingmethotrexate (MTX). This drug is a direct inhibitor of the dhfr enzymeand allows for isolation of resistant colonies which amplify their dhfrgene copy number and therefore the antibody gene sufficiently to surviveunder these conditions.

Purified antibody molecules, e.g. immunoglobulins, may be prepared by amethod comprising the steps of

-   (a) recombinantly expressing a heterologous nucleic acid molecule    encoding an antibody molecule as defined herein above in a mammalian    cell, e.g. a CHO or a HEK 293 cell; and-   (b) purifying said recombinantly expressed antibody molecule by a    method comprising the steps of    -   (b1) protein A column purification;    -   (b2) ion exchange column purification, e.g. a cation exchange        chromatography; and, optionally,    -   (b3) size exclusion column purification.

The purification protocol may comprise further steps, like furtherconcentration steps, e.g. diafiltration or analytical steps, e.g.involving analytical columns. The method/process may also comprise virusinactivation steps and/or viral removal steps e.g. viafiltrations/nano-filtrations. It is also envisaged and feasible thatparticular certain steps are repeated (e.g. two ion exchangechromatography steps may be carried out) or that certain steps (e.g.size exclusion chromatography) may be omitted.

Protein A is a group specific ligand which binds to the Fc region ofmost IgG1 isotypes. It is synthesized by some strains of Staphylococcusaureus and can be isolated therefrom and coupled to chromatographicbeads. Several types of gel preparations are available commercially.

An example for a protein A column which may be used is a MabSelect(Trademark) column. Ideally the column is equilibrated with 25 mMTris/HCl, 25 mM NaCl, 5 mM EDTA, the cell culture supernatant is loadedonto the column, the column is washed with 1 M Tris/HCl pH 7.2 and theantibody is eluted at pH 3.2 using 100 mM acetic acid.

Cation-exchange chromatography exploits interactions between positivelycharged groups in a stationary phase and the sample which is in themobile phase. When a weak cation exchanger (e.g. CM Toyopearl 650®) isused, the following chromatographic steps are performed: Afterpreequilibration with 100 mM acetic acid pH 4, loading of Protein Aeluate and washing with 100 mM acetic acid pH 4 the antibody is elutedand fractionated by applying steps of 250 mM sodium acetate (pH 7.8-8.5)and 500 mM sodium acetate (pH 7.8-8.5). With the first step a mixture ofdouble-glycosylated isoform fraction and mono-glycosylated isoformfraction are normally eluted, using the second step the non-glycosylatedisoform fraction is normally eluted.

From a strong cation exchanger (e.g. SP Toyopearl 650) the antibody canbe eluted by salt steps: After equilibration of the column with 50 mMacetic acid pH 5.0, loading the Protein A eluate with pH 4 the firstelution step using 50 mM acetic acid and 210 mM sodium chloride isperformed. Then a second elution step of 50 mM acetic acid and 350 mMsodium chloride is applied. By the first salt step a mixture of thedouble-glycosylated isoform fraction and mono-glycosylated isoformfraction are normally eluted, by the second salt step thenon-glycosylated isoform is normally eluted.

In addition the antibody may also be eluted from a strong cationexchanger column (e.g. SP-Sepharose®) by a salt gradient: Afterpreequilibration, loading and washing the column at pH 4.5 a saltgradient is applied from 50 mM MES pH 5.8 to 50 mM MES/1 M sodiumchloride pH 5.8. Here the double-glycosylated isoform, mono-glycosylatedisoform and non-glycosylated isoform fractions are normally elutedseparately. In the following double-glycosylated isoform fraction andmono-glycosylated isoform fraction may be pooled to result in theproduct pool and/or a desired antibody mixture.

Further purification of the mixture of double- and mono-glycosylatedantibody molecules, e.g. immunoglobulins, may be performed by sizeexclusion chromatography. An example of a useful column is a Superdex200® column. Examples of running buffers include histidine/sodiumchloride, e.g. 10 mM histidine/125 mM sodium chloride/pH 6, andphosphate buffered saline (PBS).

Anion exchange chromatography in the flow through mode followed by aconcentration/diafiltration is an alternative purification step. QSepharose® is an example for a resin for the anion exchange step. Forexample, the eluate from the SP chromatography may be threefold dilutedwith 37.5 mM Tris/HCl pH 7.9 and passed over a Q-Sepharose columnpre-equilibrated with 25 mM Tris/83 mM sodium acetate. The flow throughis collected, adjusted to pH 5.5 and concentrated by ultrafiltrationusing e.g. a Hydrosart 30 kD® membrane. In the following the concentratemay be diafiltrated against for example 10 volumes of 20 mMhistidine/HCl pH 5.5.

The above recited purification protocol may also comprise as anadditional step (c) an analytical chromatography step, like the use of aMono-S HR5/5 column. However, also further steps, like diafiltration,for example for concentration of the antibody molecules, is envisaged.

In one embodiment of the present invention, a composition, antibodypreparation or antibody pool is provided comprising antibody moleculesas described herein or antibody molecules as prepared by the methodprovided above. In this embodiment of the invention, said compositioncomprises mono- or double-glycosylated antibodies. In anotherembodiment, said composition comprises mono- and double-glycosylated (inthe variable region(s) of the heavy chain(s)) antibodies and saidcomposition is derived of antibody molecules which lack theglycosylation in the variable region. In context of this embodiment, theterm “antibody pool” relates to a mixture of mono- anddouble-glycosylated (in the variable region(s) of the heavy chain(s))antibodies which may have been individually isolated and then arecombined to one mixture. The antibody mixtures or antibody poolsprovided herein may comprise 50% mono-glycosylated and 50%double-glycosylated antibodies as defined herein. However, alsoenvisaged are the ratios of 30/70 to 70/30. Yet, the person skilled inthe art is aware that also other ratios are envisaged in the antibodymixtures of this invention. For example, also 10/90 or 90/10, 20/80 or80/20 as well as 40/60 or 60/40 may be employed in context of thisinvention. As also documented in the examples, a particular useful ratioin the ANTIBODY MIXTURES of the invention comprises double-glycosylatedand mono-glycosylated antibody as defined herein above is a ratio from40/60 to 45/55.

The compositions provided herein are particularly useful in diagnosticor in a pharmaceutical composition.

Accordingly, the invention provides for diagnostic or pharmaceuticalcompositions comprising

-   (a) an antibody molecule as defined above comprising one antigen    binding site with a glycosylated Asn;-   (b) an antibody molecule as defined above, comprising two antigen    binding sites with a glycosylated Asn; or, most preferably,-   (c) a combination of antibody molecules (a) to (b).

The combination (c) as provided herein, comprising the antibodymolecule(s) comprising one antigen binding site with a glycosylated Asnand the antibody molecule(s) comprising two antigen binding sites with aglycosylated Asn are devoid of non-glycosylated (in respect to thevariable region of the heavy chain) isoforms. As pointed out above, theterm “devoid of non-glycosylated (in respect to the variable region ofthe heavy chain) isoform” relates to combinations/antibodypools/antibody preparations, wherein less than 5%, e.g. less than 4%,less than 3%, less than 2%, less than 1% or even less than 0.5% of theantibody species in said combination is non-glycosylated in the variableregion of the heavy chain. As demonstrated in the examples, saidcombinations/antibody pools/antibody preparations may comprise almost no(less than 0.5%) non-glycosylated isoforms. The percentage and/or theamount of a given glycosylation isoform (as defined herein, e.g.glycosylation in the variable region of the heavy chain, see inter aliaappended FIG. 14) in a given ANTIBODY COMPOSITION may easily bedetermined by methods known in the art. These methods comprise, but arenot limited to, mass spectrometry, SDS-PAGE analysis ion exchange, HPLL,ELISA and the like.

As shown in the appended examples, the specific and sensitiveimmuno-decoration of genuine Alzheimer's β-amyloid plaques by theantibodies of the invention is demonstrated in vitro withimmunohistochemical staining experiments using cryo-sections of humanbrain tissue from Aβ patients. Effective staining of β-amyloid plaquesfrom brain slices was demonstrated also with human anti-Aβ antibodiesfrom patients vaccinated with Aβ (Hock, 2002, Nature Medicine, 8,1270-1275). Further, immuno-decoration is also demonstrated in atransgenic animal model featuring human β-amyloid plaque burden(Richards, 2003, J. Neuroscience, 23, 8989-9003). In similar animalmodels it had been demonstrated that this plaque binding led to theirclearance and subsequently to an improvement of disease relatedsymptoms, whereas the involvement of Fc-dependent processes had beendiscussed (Bard, 2000, Nature Medicine, 6, 916-919; Wilcock, 2003,Neurobiology Disease, 15, 11-20; Wilcock, 2004, J. Neuroscience, 24,6144-6151). Furthermore, effective binding of anti-Aβ antibodies toβ-amyloid plaques was reported to correlate with slower diseaseprogression (Hock, 2002, Nature Medicine, 8, 1270-1275; Hock, 2003,Neuron, 38, 547-554). This and post-mortem analysis of human braintissue suggests that phagocytosis of microglia cells is mechanisticallyinvolved in the plaque clearance in man (Nicoll, 2003, Nature Medicine,9, 448-452). Therefore the antibody of the present invention orcomprised in particular in pharmaceutical compositions is a human IgG1,which is mainly responsible for FcR-dependent processes in humans. Theefficient β-amyloid plaque immuno-decoration of the antibodies of theinvention/the mixture of the invention suggests that the drug will beefficacious for passive immunization to clear existing and preventformation of β-amyloid plaques in humans.

In addition antibodies should preferably cross the blood-brain-barrierto reach their place of destination. For large size molecules as humanIgGs this process is dramatically reduced, so that only about 0.1 to0.2% of the plasma concentration of an antibody can be reached in CSF.The mechanism of plaque clearance is still a subject of controversialdebates, which might involve peripheral effects on the Aβ peptide(Dodart, 2002, Nature Neuroscience, 5: 452-457). Thus, the generatedtherapeutic antibody or the corresponding inventive mixtures of mono-and double glycosylated (in the variable region heavy chain) of theinvention have also the property to depolymerize Aβ multimers in vitrowithout involvement of Fc-dependent processes and to bind to soluble Aβmonomers and oligomers in CSF, since neutralization of soluble monomericAβ peptides or oligomeric Aβ peptides (e.g. aggregation intermediates)may also contribute to overall amyloid lowering effect (Du, 2003, Brain,126: 1-5).

The compositions of the invention may be administered in solid or liquidform and may be, inter alia, in a form of (a) powder(s), (a) tablet(s),(a) solution(s) or (an) aerosol(s). Said composition may comprise on ormore antibodies/antibody molecules of the invention most preferably amixture of mono- and double-glycosylated antibodies as provided herein.

It is preferred that said pharmaceutical composition, optionallycomprises a pharmaceutically acceptable carrier and/or diluent. Theherein disclosed pharmaceutical composition may be particularly usefulfor the treatment of neurological and/or neurodegenerative disorders.Said disorders comprise, but are not limited to Alzheimer's disease,amyothrophic lateral sclerosis (ALS), hereditary cerebral hemorrhagewith amyloidosis Dutch type, Down's syndrome, HIV-dementia, Parkinson'sdisease and neuronal disorders related to aging. The pharmaceuticalcomposition of the invention is, inter alia, envisaged as potentinhibitor of amyloid plaque formation or as a potent stimulator for thede-polymerization of amyloid plaques. Therefore, the present inventionprovides for pharmaceutical compositions comprising the compounds of theinvention to be used for the treatment of amyloidogenicdiseases/disorders. The term “amyloidogenic disease/disorder” includesany disease associated with or caused by the formation or deposition ofamyloid fibrils and/or pathological APP proteolysis. Exemplaryamyloidogenic disease include, but are not limited to Alzheimer'sdisease (AD), Down's Syndrome, dementia associated with Lewy bodyformation, Parkinson's Disease with dementia, mild cognitive impairment,cerebral amyloid angiopathy and vascular dementia. Differentamyloidogenic diseases are defined and/or characterized by the nature ofthe polypeptide-component of the amyloid deposits. For example, theamyloid-β protein is characteristic for the amyloid deposits found insubjects having Alzheimer's disease.

Examples of suitable pharmaceutical carriers, excipients and/or diluentsare well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Compositions comprising suchcarriers can be formulated by well known conventional methods. Suitablecarriers may comprise any material which, when combined with the anti-Aβspecific binding agent or antibody, retains the high-affinity binding ofAβ and is nonreactive with the subject's immune systems includingexcipients, surfactants, tonicity agents and the like; see Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. Administration of the suitable compositions may beeffected by different ways, e.g., by parenteral, subcutaneous,intraperitoneal, topical, intrabronchial, intrapulmonary and intranasaladministration and, if desired for local treatment, intralesionaladministration. Parenteral administrations include intraperitoneal,intramuscular, intradermal, subcutaneous intravenous or intraarterial,administration. It is particularly preferred that said administration iscarried out by injection and/or delivery, e.g., to a site in a brainartery or directly into brain tissue. The compositions of the inventionmay also be administered directly to the target site, e.g., by biolisticdelivery to an external or internal target site, like the brain.

Pharmaceutical compositions comprising the herein described glycosylatedantibodies are prepared by mixing the antibody having the desired degreeof purity with optional physiologically acceptable carriers, excipients,stabilizers, surfactants, buffers and/or tonicity agents. Acceptablecarriers, excipients and/or stabilizers are nontoxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid, glutathione, cysteine, methionine and citric acid;preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol,p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride orcombinations thereof); amino acids such as arginine, glycine, ornithine,lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine,alanine, phenylalanine, tyrosine, tryptophan, methionine, serine,proline and combinations thereof; monosaccharides, disaccharides andother carbohydrates; low molecular weight (less than about 10 residues)polypeptides; proteins, such as gelatin or serum albumin; chelatingagents such as EDTA; sugars such as trehalose, sucrose, lactose,glucose, mannose, maltose, galactose, fructose, sorbose, raffinose,glucosamine, N-Methylglucosamine (so-called “Meglumine”), galactosamineand neuraminic acid; and/or non-ionic surfactants such as Tween, BrijPluronics, Triton-X or polyethylene glycol (PEG).

The pharmaceutical composition may be in a liquid form, a lyophilizedform or a liquid form reconstituted from a lyophilized form, wherein thelyophilized preparation is to be reconstituted with a sterile solutionprior to administration. The standard procedure for reconstituting alyophilized composition is to add back a volume of pure water (typicallyequivalent to the volume removed during lyophilization), however alsosolutions comprising antibacterial agents may be used for the productionof pharmaceutical compositions for parenteral administration; see alsoChen (1992) Drug Dev Ind Pharm 18, 1311-54.

Exemplary antibody concentrations in the pharmaceutical composition mayrange from about 1 mg/mL to about 200 mg/ml or from about 50 mg/mL toabout 200 mg/mL, or from about 150 mg/mL to about 200 mg/mL. For clarityreasons, it is emphasized that the concentrations as indicated hereinrelate to the concentration in a liquid or in a liquid that isaccurately reconstituted from a solid form.

An aqueous formulation of the antibody may be prepared in a pH-bufferedsolution, e.g., at pH ranging from about 4.0 to about 7.0, or from about5.0 to about 6.0, or alternatively about 5.5. Examples of buffers thatare suitable for a pH within this range include phosphate-, histidine-,citrate-, succinate-, acetate-buffers and other organic acid buffers.The buffer concentration can be from about 1 mM to about 100 mM, or fromabout 5 mM to about 50 mM, depending, e.g., on the buffer and thedesired tonicity of the formulation.

A tonicity agent may be included in the antibody formulation to modulatethe tonicity of the formulation. Exemplary tonicity agents includesodium chloride, potassium chloride, glycerin and any component from thegroup of amino acids, sugars as well as combinations thereof. Preferablythe aqueous formulation is isotonic, although hypertonic or hypotonicsolutions may be suitable. The term “isotonic” denotes a solution havingthe same tonicity as some other solution with which it is compared, suchas physiological salt solution and the blood serum. Tonicity agents maybe used in an amount of about 5 mM to about 350 mM, in particular in anamount of 105 mM to 305 nM.

A surfactant may also be added to the antibody formulation to reduceaggregation of the formulated antibody and/or minimize the formation ofparticulates in the formulation and/or reduce adsorption. Exemplarysurfactants include polyoxyethylensorbitan fatty acid esters (Tween),polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers(Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer,Pluronic), and sodium dodecyl sulphate (SDS). Preferredpolyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (soldunder the trademark Tween 20™) and polysorbate 80 (sold under thetrademark Tween 80™). Preferred polyethylene-polypropylene copolymersare those sold under the names Pluronic® F68 or Poloxamer 188™.Preferred Polyoxyethylene alkyl ethers are those sold under thetrademark Brij™. Exemplary concentrations of surfactant may range fromabout 0.001% to about 1% w/v.

A lyoprotectant may also be added in order to protect the labile activeingredient (e.g. a protein) against destabilizing conditions during thelyophilization process. For example, known lyoprotectants include sugars(including glucose and sucrose); polyols (including mannitol, sorbitoland glycerol); and amino acids (including alanine, glycine and glutamicacid). Lyoprotectants are generally used in an amount of about 10 mM to500 nM.

In one embodiment, the formulation contains the above-identified agents(i.e. glycosylated antibody, surfactant, buffer, stabilizer and/ortonicity agent) and is essentially free of one or more preservatives,such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol,methyl or propyl parabens, benzalkonium chloride, and combinationsthereof. In another embodiment, a preservative may be included in theformulation, e.g., at concentrations ranging from about 0.001 to about2% (w/v).

In one embodiment, the antibody formulation of the invention is a liquidor lyophilized formulation suitable for parenteral administration thatmay comprise:

-   -   about 1 to about 200 mg/mL of the herein described glycosylated        antibodies or ANTIBODY COMPOSITION,    -   about 0.001 to about 1% of at least one surfactant;    -   about 1 to about 100 mM of a buffer;    -   optionally about 10 to about 500 mM of a stabilizer and/or about        5 to about 305 mM of a tonicity agent;    -   at a pH of about 4.0 to about 7.0.

In a preferred embodiment, the parenteral formulation of the inventionis a liquid or lyophilized formulation comprising:

-   -   about 1 to about 200 mg/mL of the herein described glycosylated        antibodies or ANTIBODY COMPOSITION,    -   0.04% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Sucrose,    -   at pH 5.5.

In a more preferred embodiment, the parenteral formulation according tothe invention also comprises a lyophilized formulation comprising:

-   -   15 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.04% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Sucrose,    -   at pH 5.5;        or    -   75 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.04% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Sucrose,    -   at pH 5.5;        or    -   75 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Sucrose,    -   at pH 5.5;        or    -   75 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.04% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Trehalose,    -   at pH 5.5;        or    -   75 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Trehalose,    -   at pH 5.5

In another more preferred embodiment, the parenteral formulationaccording to the invention also comprises a liquid formulationcomprising:

-   -   7.5 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.022% Tween 20 w/v,    -   120 mM L-histidine,    -   250 125 mM Sucrose,    -   at pH 5.5;        or    -   37.5 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   10 mM L-histidine,    -   125 mM Sucrose,    -   at pH 5.5;        or    -   37.5 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.01% Tween 20 w/v,    -   10 mM L-histidine,    -   125 mM Sucrose,    -   at pH 5.5;        or    -   37.5 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   10 mM L-histidine,    -   125 mM Trehalose,    -   at pH 5.5;        or    -   37.5 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.01% Tween 20 w/v,    -   10 mM L-histidine,    -   125 mM Trehalose,    -   at pH 5.5;        or    -   75 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Trehalose,    -   at pH 5.5;        or    -   75 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Mannitol,    -   at pH 5.5;        or    -   75 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   20 mM L histidine,    -   140 mM Sodium chloride,    -   at pH 5.5;        or    -   150 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Trehalose,    -   at pH 5.5.        or    -   150 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Mannitol,    -   at pH 5.5.        or    -   150 mg/mL of the herein described glycosylated antibodies or        ANTIBODY COMPOSITION,    -   0.02% Tween 20 w/v,    -   20 mM L-histidine,    -   140 mM Sodium chloride,    -   at pH 5.5.        or    -   10 mg/mL Abeta antibody,    -   0.01% Tween 20 w/v,    -   20 mM L-histidine,    -   140 mM Sodium chloride,        at pH 5.5.

In one embodiment, the pharmaceutical composition of the presentinvention is the liquid formulation which comprises:

-   -   10 mg/mL Abeta antibody,    -   0.01% Tween 20 w/v,    -   20 mM L-histidine,    -   140 mM Sodium chloride,        at pH 5.5.

In another embodiment, the pharmaceutical composition of the presentinvention is the lyophilized formulation which comprises:

-   -   75 mg/mL Abeta antibody,    -   0.04% Tween 20 w/v,    -   20 mM L-histidine,    -   250 mM Sucrose,        at pH 5.5.

The term “herein described glycosylated antibodies” in context ofexemplified formulations may comprise in this invention the hereindefined mono-glycosylated antibodies, the herein defineddouble-glycosylated antibodies as well as mixtures thereof.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depend upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. Proteinaceous pharmaceutically active mattermay be present in amounts between 1 ng and 20 mg/kg body weight perdose, e.g. between 0.1 mg to 10 mg/kg body weight, e.g. between 0.5 mgto 5 mg/kg body weight; however, doses below or above this exemplaryrange are envisioned, especially considering the aforementioned factors.If the regimen is a continuous infusion, it should also be in the rangeof 1 μg to 10 mg per kilogram of body weight per minute.

The pharmaceutical compositions as described herein may be formulated tobe short-acting, fast-releasing, long-acting, or sustained-releasing.Hence, the pharmaceutical compositions may also be suitable for slowrelease or for controlled release.

Sustained-release preparations may be prepared using methods well knownin the art. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody in which the matrices are in the form of shaped articles, e.g.films or microcapsules. Examples of sustained-release matrices includepolyesters, copolymers of L-glutamic acid and ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, hydrogels, polylactides,degradable lactic acid-glycolic acid copolymers andpoly-D-(−)-3-hydroxybutyric acid. Possible loss of biological activityand possible changes in immunogenicity of antibodies comprised insustained-release preparations may be prevented by using appropriateadditives, by controlling moisture content and by developing specificpolymer matrix compositions.

Progress can be monitored by periodic assessment. The compositions, i.e.the mono- and/or double-glycosylated antibodies of the invention or amixture thereof, may be administered locally or systemically. It is ofnote that peripherally administered antibodies can enter the centralnervous system, see, inter alia, Bard (2000), Nature Med. 6, 916-919.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents depending on the intended use ofthe pharmaceutical composition. Said agents may be drugs acting on thecentral nervous system, like, neuroprotective factors, cholinesteraseinhibitors, agonists of Ml muscarinic receptor, hormones, antioxidants,inhibitors of inflammation etc. It is particularly preferred that saidpharmaceutical composition comprises further agents like, e.g.neurotransmitters and/or substitution molecules for neurotransmitters,vitamin E or alpha-lipoic acid.

The person skilled in the art, in particular but not limitingbiochemists, biologists, chemists, pharmacists and groups of saidprofessionals are readily in a position to work and generate the aboverecited pharmaceutical compositions. Also medical personal skilled inthey art, like attending physicians are aware how such pharmaceuticalcompositions may be administered to a patient in need of a treatmentwith the herein defined pharmaceutical compositions. Such anadministration may comprise systemic administration, e.g. via infusionsand/or injections. However, also the direct administration of thecompounds and/or compound mixtures of the invention to the brain isenvisaged. For example, the compound or compound mixture or compoundformulation may be administered by direct intraventricular orintrathecal injection to the brain, preferably via slow infusion tominimize impact on brain parenchyma. Also slow release implant in thebrain may be employed. It is also envisaged that gene therapy approachesare employed, for example by use of implanted recombinant cells thatproduce the antibodies as defined in this invention. These “recombinantcells” should be capable of providing the herein defined glycosylationsin the variable regions/parts of the antibodies described herein, inparticular the anti-Aβ antibodies of the invention. Yet, as pointed outabove one advantage of the antibodies/antibody mixtures of the presentinvention is their capability to cross the blood-brain barrier and tobind to amyloid plaques. The pharmaceutical compositions of theinvention described infra can be used for the treatment of all kinds ofdiseases hitherto unknown or being related to or dependent onpathological APP aggregation or pathological APP processing. They may beparticularly useful for the treatment of Alzheimer's disease and otherdiseases where extracellular deposits of amyloid-β, appear to play arole. They may be desirably employed in humans, although animaltreatment is also encompassed by the methods, uses and compositionsdescribed herein.

In a preferred embodiment of the invention, the composition of thepresent invention as disclosed herein above is a diagnostic compositionfurther comprising, optionally, suitable means for detection. Thediagnostic composition comprises at least one of the aforementionedcompounds of the invention, namely the glycosylated antibodies describedherein.

Said diagnostic composition may comprise the compounds of the invention,in particular the glycosylated antibody molecules of the presentinvention, soluble form/liquid phase but it is also envisaged that saidcompounds are bound to/attached to and/or linked to a solid support.

Solid supports may be used in combination with the diagnosticcomposition as defined herein or the compounds of the present inventionmay be directly bound to said solid supports. Such supports are wellknown in the art and comprise, inter alia, commercially available columnmaterials, polystyrene beads, latex beads, magnetic beads, colloid metalparticles, glass and/or silicon chips and surfaces, nitrocellulosestrips, membranes, sheets, duracytes, wells and walls of reaction trays,plastic tubes etc. The compound(s) of the invention, in particular theantibodies of the present invention, may be bound to many differentcarriers. Examples of well-known carriers include glass, polystyrene,polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran,nylon, amyloses, natural and modified celluloses, polyacrylamides,agaroses, and magnetite. The nature of the carrier can be either solubleor insoluble for the purposes of the invention. Appropriate labels andmethods for labeling have been identified above and are furthermorementioned herein below. Suitable methods for fixing/immobilizing saidcompound(s) of the invention are well known and include, but are notlimited to ionic, hydrophobic, covalent interactions and the like.

It is particularly preferred that the diagnostic composition of theinvention is employed for the detection and/or quantification of APPand/or APP-processing products, like amyloid-β or for the detectionand/or quantification of pathological and/or (genetically) modifiedAPP-cleavage sides.

As illustrated in the appended examples, the inventive glycosylatedantibody molecules are particularly useful as diagnostic reagents in thedetection of genuine human amyloid plaques in brain sections ofAlzheimer's Disease patients by indirect immunofluorescence.

It is preferred that said compounds of the present invention to beemployed in a diagnostic composition are detectably labeled. A varietyof techniques are available for labeling biomolecules, are well known tothe person skilled in the art and are considered to be within the scopeof the present invention. There are many different labels and methods oflabeling known to those of ordinary skill in the art. Examples of thetypes of labels which can be used in the present invention includeenzymes, radioisotopes, colloidal metals, fluorescent compounds,chemiluminescent compounds, and bioluminescent compounds.

Commonly used labels comprise, inter alia, fluorochromes (likefluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radishperoxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes(like ³²P or ¹²⁵I), biotin, digoxygenin, colloidal metals, chemi- orbioluminescent compounds (like dioxetanes, luminol or acridiniums).Labeling procedures, like covalent coupling of enzymes or biotinylgroups, iodinations, phosphorylations, biotinylations, etc. are wellknown in the art.

Detection methods comprise, but are not limited to, autoradiography,fluorescence microscopy, direct and indirect enzymatic reactions, etc.Commonly used detection assays comprise radioisotopic ornon-radioisotopic methods. These comprise, inter alia, Westernblotting,overlay-assays, RIA (Radioimmuno Assay) and IRMA (ImmuneRadioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (EnzymeLinked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay), and CLIA(Chemioluminescent Immune Assay).

Furthermore, the present invention provides for the use of theglycosylated antibody molecules of invention, or an antibody moleculeproduced by the method of the invention, or a mixture of mono- anddouble-glycosylation antibodies as provided herein for the preparationof a pharmaceutical or a diagnostic composition for the prevention,treatment and/or diagnosis of a disease associated with amyloidogenesisand/or amyloid-plaque formation. It is further preferred that thecompounds described herein, in particular the antibody molecules of theinvention, be employed in the prevention and/or treatment ofneuropathologies associated with modified or abnormal APP-processingand/or amyloidogenesis. The antibody molecules, e.g. in the format of(engineered) immunoglobulins, like antibodies in a IgG framework, inparticular in an IgG1-framework, or in the format of chimeric antibodies(in particular fully humanized antibodies or complete antibodies),bispecific antibodies, single chain Fvs (scFvs) or bispecific scFvs andthe like are employed in the preparation of the pharmaceuticalcompositions provided herein. Yet, the antibody molecules and mixturesprovided herein are also useful in diagnostic settings as documented inthe appended examples, since the antibody molecules of the inventionspecifically interact with/detect Aβ4 and/or amyloid deposits/plaques.

Therefore an inventive use of the compounds of the present invention isthe use for the preparation of a pharmaceutical composition for aneurological disorder which calls for amelioration, for example bydisintegration of β-amyloid plaques, by amyloid (plaque) clearance or bypassive immunization against β-amyloid plaque formation. As illustratedin the appended examples, the inventive antibody molecules areparticularly useful in preventing Aβ aggregation and inde-polymerization of already formed amyloid aggregates. Accordingly, theinventive glycosylated antibodies or a mixture of mono- anddouble-glycosylated antibodies as described herein are to be employed inthe reduction of pathological amyloid deposits/plaques, in the clearanceof amyloid plaques/plaque precursors as well as in neuronal protection.It is in particular envisaged that the antibody molecules of theinvention be employed in the in vivo prevention of amyloid plaques aswell as in in vivo clearance of pre-existing amyloid plaques/deposits.Furthermore, the antibody molecules or the mixtures of the invention maybe employed in passive immunization approaches against Aβ peptide andaggregates of Aβ, namely amyloid-β plaques. Clearance of Aβ4/Aβ4deposits may, inter alia, be achieved by the medical use of antibodiesof the present invention which comprise an Fc-part. Said Fc-part of anantibody may be particularly useful in Fc-receptor mediated immuneresponses, e.g. the attraction of macrophages (phagocytic cells and/ormicroglia) and/or helper cells. For the mediation of Fc-part-relatedimmunoresponses, the antibody molecule of the invention is preferably inan (human) IgG1-framework. As discussed herein, the preferred subject tobe treated with the inventive antibody molecules, or antibody mixturesis a human subject. Other frameworks, like IgG2a- or IgG2b-frameworksfor the inventive antibody molecules are also envisaged. Immunoglobulinframeworks in IgG2a and IgG2b format are particular envisaged in mousesettings, for example in scientific uses of the inventive antibodymolecules, e.g. in tests on transgenic mice expressing (human) wild typeor mutated APP, APP-fragments and/or Aβ4.

The above recited diseases associated with amyloidogenesis and/oramyloid-plaque formation comprise, but are not limited to dementia,Alzheimer's disease, motor neuropathy, Parkinson's disease, ALS(amyotrophic lateral sclerosis), scrapie, HIV-related dementia as wellas Creutzfeld-Jakob disease, hereditary cerebral hemorrhage, withamyloidosis Dutch type, Down's syndrome and neuronal disorders relatedto aging. The antibody molecules of the invention and the compositionsprovided herein may also be useful in the amelioration and or preventionof inflammatory processes relating to amyloidogenesis and/or amyloidplaque formation.

Accordingly, the present invention also provides for a method fortreating, preventing and/or delaying neurological and/orneurodegenerative disorders comprising the step of administering to asubject suffering from said neurological and/or neurodegenerativedisorder and/or to a subject susceptible to said neurological and/orneurodegenerative disorder an effective amount of a an anti Aβ antibodymolecule or a mixture of the inventive mono- and/or double-glycosylatedA-beta antibodies as provided herein and/or a composition as definedherein above. The treatment as provided herein may comprise theadministration for the compounds/compositions of this invention alone orin form of a co-therapy treatment, i.e. in combination with other drugsor medicaments. In a particular preferred embodiment of the invention, amethod for treating, preventing and/or delaying neurological and/orneurodegenerative disorders is provided that comprises the step ofadministering to a patient in need of a corresponding medicalintervention the antibody mixture comprising mono- anddouble-glycosylated antibodies directed against Aβ and as providedherein.

The term “treatment” used herein envisages the administration of mono-and/or double-glycosylated antibodies (or mixtures thereof) as describedherein to a patient in need thereof. Said patient may be a humanpatient, in one embodiment an human suffering from or being susceptibleto a disorder related to pathological APP processing. Accordingly, theterm “treatment” as used herein comprises the prophylactic as well asthe curative administration of the compounds or compound mixturesprovided herein.

An disorder to be treated by the compounds and composition providedherein is Alzheimer's disease. Patients having a diagnosis of probableAlzheimer's disease based on the National Institute of Neurological andCommunicative Disorders and Stroke/Alzheimer's Disease and RelatedDisorders Association criteria for this diagnosis (NINCDS/ADRDAcriteria) Mckhann et al., 1984.

Also envisaged in context of this invention is the medical use of thecompounds and/or compositions provided herein in a “co-therapy” setting.for example in the case of APP-related disorders, like Alzheimer'sdisease. In said case, co-therapy with approved medicaments, likememantine, doneprezil, rivastigmine or galantamine, is envisaged.

In yet another embodiment, the present invention provides for a kitcomprising at least one glycosylated antibody molecule as defined hereinor the mixture of the inventive mono- and/or double-glycosylated methodsas provided herein. Advantageously, the kit of the present inventionfurther comprises, optionally (a) buffer(s), storage solutions and/orremaining reagents or materials required for the conduct of medical,scientific or diagnostic assays and purposes. Furthermore, parts of thekit of the invention can be packaged individually in vials or bottles orin combination in containers or multicontainer units.

The kit of the present invention may be advantageously used, inter alia,for carrying out the method of the invention and could be employed in avariety of applications referred herein, e.g., as diagnostic kits, asresearch tools or medical tools. Additionally, the kit of the inventionmay contain means for detection suitable for scientific, medical and/ordiagnostic purposes. The manufacture of the kits follows preferablystandard procedures which are known to the person skilled in the alt.

The figures show:

FIG. 1: Plasmid map showing the insertion sites for the heavy and lightchain sequences

FIG. 2: Example of an analytical chromatogram

FIG. 3: Chromatogram of a CMT column as described in the text.Double-glycosylated and mono-glycosylated isoforms are eluting in doublepeak 1, the non-glycosylated isoform is eluting in peak 2

FIG. 4: Whole IgG ESI-MS analysis of ANTIBODY A isoforms. Molecular massof main peak is indicated in Da. A: non-glycosylated ANTIBODY A; B:mono-glycosylated ANTIBODY A; C: double-glycosylated ANTIBODY A

FIG. 5: Scheme of deduced ANTIBODY N-glycosylation patterns. Structuresthat occur only partially are indicated by parenthesis. A: Complex Type;B: Hybrid Type; C: Oligomannose Type; GlcNAc=N-acetyl-glucosamine,Man=mannose; Gal=galactose; Fuc=fucose; NeuAc=N-acetyl-neuraminic acid

FIG. 6: Schematic presentation of carbohydrate structures at Asn306 ofANTIBODY A deduced from MS and HPAEC-PAD analysis. Structures that occuronly partially are indicated by parenthesis.GlcNAc=N-acetyl-glucosamine, Man=mannose; Gal=galactose; Fuc=fucose;NeuAc=N-acetyl-neuraminic acid

FIG. 7: Binding of ANTIBODY A isoforms to immobilized fibrillar Aβ40(Biacore sensor chip). Antibody concentration 60 nM. Binding curve of amixture of all isoforms, i.e. before purification is also shown asindicated.

FIG. 8: Epitope mapping of ANTIBODY A COMPOSITION by pepspot analysis.A) pepspot signals of indicated single overlapping decapeptide spots; B)densitometric analysis of signal intensity of single overlappingdecapeptide spots.

FIG. 9: De-polymerization Assay. ANTIBODY A COMPOSITION and ANTIBODY Aisoforms induce release of biotinylated Aβ from aggregated Aβ

FIG. 10: ANTIBODY A COMPOSITION and comprising ANTIBODY A isoformscapture soluble Aβ from human cerebrospinal fluid (CSF). Average of 4CSF samples from Alzheimer's disease patients analyzed in pools of 2.Two immunoprecipitations followed by Western blots per pool withquantification of captured Aβ by densitometry of Western blots. Thehighest Aβ value on a given series of Western blots was taken as 100%

FIG. 11: Indirect immunofluorescence staining of human amyloid plaqueswith ANTIBODY A isoforms in vitro. Highly sensitive and specificdetection of genuine ex vivo human β-amyloid plaques after staining with10 ng/ml ANTIBODY A concentration. Bound ANTIBODY A was revealed by goatanti-human (H+L)-Cy3 for (A) ANTIBODY A COMPOSITION; (B)double-glycosylated ANTIBODY A; (C) mono-glycosylated ANTIBODY A; and(D) non-glycosylated ANTIBODY A. Scale bar=80 μm

FIG. 12: In vivo immuno-decoration of PS2APP transgenic mouse plaqueswith glycosylated ANTIBODY A isoforms revealed by confocal microscopy.Immunodecoration reveals in vivo binding of ANTIBODY A isoforms 3 daysafter a single dose of 1 mg of ANTIBODY A isoforms. Representativeimages of the distribution of ANTIBODY A isoforms are shown for thedouble- (A), mono- (B), and non-glycosylated (C) ANTIBODY A isoform.Scale bar=80 μm

FIG. 13: Binding analysis of anti-Aβ antibodies to cell surface APP.Antibody binding to human APP-transfected HEK293 cells andnon-transfected control cells analyzed by flow cytometry.

FIG. 14: Scheme of ANTIBODY A non-, mono- and double-glycosylatedantibody molecules (immunoglobulins).

FIG. 15: Total plaque surface (A), total plaque number (B) and plaquenumber and size distribution (C) in the thalamus region after 5 monthtreatment with ANTIBODY A COMPOSITION (which comprises mono- anddouble-glycosylated ANTIBODY A), double-glycosylated andmono-glycosylated ANTIBODY A isoforms (20 mg/kg weekly, i.v.) orvehicle.

FIG. 16: Total plaque surface (A), total plaque number (B) and plaquenumber and size distribution (C) in the cortex and corpus callosumregion after 5 month treatment with ANTIBODY A COMPOSITION (whichcomprises mono- and double-glycosylated ANTIBODY A), double-glycosylatedand mono-glycosylated ANTIBODY A isoforms (20 mg/kg weekly, i.v.) orvehicle.

FIG. 17: Total plaque surface (A), total plaque number (B) and plaquenumber and size distribution (C) in the hippocampus region after 5 monthtreatment with ANTIBODY A COMPOSITION (which comprises mono- anddouble-glycosylated ANTIBODY A), double-glycosylated andmono-glycosylated ANTIBODY A isoforms (20 mg/kg weekly, i.v.) orvehicle.

FIG. 18: Total plaque surface (A), total plaque number (B) and plaquenumber and size distribution (C) in the subiculum region after 5 monthtreatment with ANTIBODY A COMPOSITION (which comprises mono- anddouble-glycosylated ANTIBODY A), double-glycosylated andmono-glycosylated ANTIBODY A isoforms (20 mg/kg weekly, i.v.) orvehicle.

FIG. 19: Measurement of fluorescence intensity of immunostained ANTIBODYA COMPOSITION bound to amyloid-β plaques after biweekly dosing of 0.1mg/kg with 1, 2 and 4 i.v. applications to PS2APP mice. Analysis wasdone at 2 weeks after last injection.

FIG. 20: Measurement of fluorescence intensity of immunostained ANTIBODYA COMPOSITION bound to amyloid-β plaques after monthly dosing of 0.15mg/kg with 2 and 3 i.v. applications to PS2APP mice. Analysis was doneat 2 weeks after last injection.

FIG. 21: Measurement of fluorescence intensity of immunostained ANTIBODYA COMPOSITION bound to amyloid-β plaques after 4 biweekly injections of0.05, 0.1 and 0.30 mg/kg to PS2APP mice, suggesting dose-relatedamyloid-plaque binding. Analysis was done at 2 weeks after lastinjection.

FIG. 22: Measurement of fluorescence intensity of immunostained ANTIBODYA COMPOSITION bound to amyloid-β plaques after 3 monthly injections of0.075, 0.15 and 0.45 mg/kg to PS2APP mice, suggesting dose-relatedamyloid-plaque binding. Analysis was done at 2 weeks after lastinjection.

FIG. 23: Human AD brain sections stained against Aβ with anti-Aβ murinemonoclonal antibody (BAP-2) after 40 hours incubation with ANTIBODY ACOMPOSITION at indicated concentrations together with livingdifferentiated primary human macrophages (0.8 million cells/ml). Resultsshow reduction in amyloid load indicative for antigen-dependent cellularphagocytosis effect of ANTIBODY A COMPOSITION on amyloid-β plaques.Scale bar=300 μm.

FIG. 24: Dose response of ANTIBODY A COMPOSITION on amyloid-β plaquesfrom human AD brain sections when incubated with 0.8 million cells/ml.(A) shows total plaque area and (B) staining intensity.

FIG. 25: Fluorescent microscopy of P388D1 cells incubated with 0, 0.1, 1and 10 μg/ml ANTIBODY A COMPOSITION (A to D, respectively).

FIG. 26: Quantitative measurement of dose response of ANTIBODY ACOMPOSITION using Aβ conjugated fluorobeads and P3881D1 cells (shown inrelative fluorescent units, RFU). Two independent experiments indicate aconsiderable range of efficacy for ANTIBODY A COMPOSITION.

FIG. 27: Table showing different glycan structures of ANTIBODY A in theconstant region of the heavy chain (Asn 306; first two columns) and inthe variable region of the heavy chain (Asn 52; third and fourthcolumn).

EXAMPLES

The following, non-limiting examples illustrate the invention.

Example 1 Generation of ANTIBODY A Via Cloning Techniques

In accordance with the present invention, an IgG1 molecule was generatedvia common cloning techniques. ANTIBODY A is, in its coding sequence andin its expressed amino acid sequence characterized by its variableregion of the heavy chain (V_(H)). The corresponding example of a heavyencoded by a DNA sequence as follows:

(SEQ ID NO: 5) caggtggaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcctccggatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagcgctattaatgcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaaacaccctgtatctgcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggtaatactcataagccttatggttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccagatatcgtgcgatatcgtgcaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga.and codes for the following immunoglobulin H-chain:

(SEQ ID NO: 6) QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKPNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPTEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

The same heavy chain may also be encoded by a sequence comprising anadditional “leader sequence” as shown in the following sequence

(SEQ ID NO: 25) atgaaacacctgtggttcttcctcctgctggtggcagctcccagatgggtcctgtcccaggtggaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcctccggatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagcgctattaatgcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaaacaccctgtatctgcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggtaatactcataagccttatggttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

The corresponding amino acid sequence would be

(SEQ ID NO: 26) MKHLWFFLLLVAAPRWVLSQVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Similarly, the light chain of ANTIBODY A is encoded by the followingnucleotide sequence:

(SEQ ID NO: 7) gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcgagccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcgcgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcagcctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttgaaattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagand codes for the following amino acid sequence (L-chain):

(SEQ ID NO: 8) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

Again, also here, a “leader sequence” may be employed and thecorresponding sequences would be

(SEQ ID NO: 27) atggtgttgcagacccaggtcttcatttctctgttgctctggatctctggtgcctacggggatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcgagccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcgcgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcagcctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttgaaattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgttag

This sequence encodes the following amino acid sequence

(SEQ ID NO: 28) MVLQTQVFISLLLWISGAYGDIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

These sequences above are known from MAB31 as disclosed in WO 03/070760.

However, the heavy and light chains of the exemplified ANTIBODY A mayalso be encoded by a sequence as shown below:

-   -   a) the heavy chain

(SEQ ID NO: 23) atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgattcatggagaaatagagagactgagtgtgagtgaacatgagtgagaaaaactggatttgtgtggcattttctgataacggtgtccttctgtttgcaggtgtccagtgtcaggtggagctggtggagtctgggggaggcctggtccagcctggggggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatgccatgagctgggtccgccaggctccaggcaaggggctcgagtgggtgtccgccataaacgccagcggtacccgcacctactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagaggcaaggggaacacccacaagccctacggctacgtacgctactttgacgtgtggggccaaggaaccctggtcaccgtctcctcaggtgagtcctcacaacctctctcctgcggccgcagcttgaagtctgaggcagaatcttgtccagggtctatcggactcttgtgagaattaggggctgacagttgatggtgacaatttcagggtcagtgactgtctggtttctctgaggtgagactggaatataggtcaccttgaagactaaagaggggtccaggggcttttctgcacaggcagggaacagaatgtggaacaatgacttgaatggttgattcttgtgtgacaccaagaattggcataatgtctgagttgcccaagggtgatcttagctagactctggggtttttgtcgggtacagaggaaaaacccactattgtgattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcaggtaagaatggcctctccaggtctttatttttaacctttgttatggagttttctgagcattgcagactaatcttggatatttgccctgagggagccggctgagagaagttgggaaataaatctgtctagggatctcagagcctttaggacagattatctccacatctttgaaaaactaagaatctgtgtgatggtgttggtggagtccctggatgatgggatagggactttggaggctcatttgagggagatgctaaaacaatcctatggctggagggatagttggggctgtagttggagattttcagtttttagaatgaagtattagctgcaatacttcaaggaccacctctgtgacaaccattttatacagtatccaggcatagggacaaaaagtggagtggggcactttctttagatttgtgaggaatgttccacactagattgtttaaaacttcatttgttggaaggagctgtcttagtgattgagtcaagggagaaaggcatctagcctcggtctcaaaagggtagttgctgtctagagaggtctggtggagcctgcaaaagtccagctttcaaaggaacacagaagtatgtgtatggaatattagaagatgttgcttttactcttaagttggttcctaggaaaaatagttaaatactgtgactttaaaatgtgagagggttttcaagtactcatttttttaaatgtccaaaatttttgtcaatcaatttgaggtcttgtttgtgtagaactgacattacttaaagtttaaccgaggaatgggagtgaggctctctcataccctattcagaactgacttttaacaataataaattaagtttaaaatatttttaaatgaattgagcaatgttgagttgagtcaagatggccgatcagaaccggaacacctgcagcagctggcaggaagcaggtcatgtggcaaggctatttggggaagggaaaataaaaccactaggtaaacttgtagctgtggtttgaagaagtggttttgaaacactctgtccagccccaccaaaccgaaagtccaggctgagcaaaacaccacctgggtaatttgcatttctaaaataagttgaggattcagccgaaactggagaggtcctcttttaacttattgagttcaaccttttaattttagcttgagtagttctagtttccccaaacttaagtttatcgacttctaaaatgtatttagaattcgagctcggtacagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagcaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgcctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttggtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggagcctctgcccgccccactcatgctcagggagagggtcttctggctttttcccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccaggtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaggtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggcaaatga

-   -   b) the light chain

(SEQ ID NO: 24) atggacatgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtaaggatggagaacactagcagtttactcagcccagggtgctcagtactgctttactattcagggaaattctcttacaacatgattaattgtgtggacatttgtttttatgtttccaatctcaggcgccagatgtgatatcgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgccgggccagtcagagtgttagcagcagctacttagcctggtaccagcagaaacctggccaggcgcccaggctcctcatctatggcgcatccagcagggccactggcgtgccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctggagcctgaagatttcgcgacctattactgtctgcagatttacaacatgcctatcacgttcggccaagggaccaaggtggaaatcaaacgtgagtagaatttaaactttgcggccgcctagacgtttaagtgggagatttggaggggatgaggaatgaaggaacttcaggatagaaaagggctgaagtcaagttcagctcctaaaatggatgtgggagcaaactttgaagataaactgaatgacccagaggatgaaacagcgcagatcaaagaggggcctggagctctgagaagagaaggagactcatccgtgttgagtttccacaagtactgtcttgagttttgcaataaaagtgggatagcagagttgagtgagccgtaggctgagttctctcttttgtctcctaagtttttatgactacaaaaatcagtagtatgtcctgaaataatcattaagctgtttgaaagtatgactgcttgccatgtagataccatgtcttgctgaatgatcagaagaggtgtgactcttattctaaaatttgtcacaaaatgtcaaaatgagagactctgtaggaacgagtccttgacagacagctcaaggggtttttttcctttgtctcatttctacatgaaagtaaatttgaaatgatcttttttattataagagtagaaatacagttgggtttgaactatatgttttaatggccacggttttgtaagacatttggtcctttgttttcccagttattactcgattgtaattttatatcgccagcaatggactgaaacggtccgcaacctcttctttacaactgggtgacctcgcggctgtgccagccatttggcgttcaccctgccgctaagggccatgtgaacccccgcggtagcatcccttgctccgcgtggaccactttcctgaggcacagtgataggaacagagccactaatctgaagagaacagagatgtgacagactacactaatgtgagaaaaacaaggaaagggtgacttattggagatttcagaaataaaatgcatttattattatattcccttattttaattttctattagggaattagaaagggcataaactgctttatccagtgttatattaaaagcttaatgtatataatcttttagaggtaaaatctacagccagcaaaagtcatggtaaatattctttgactgaactctcactaaactcctctaaattatatgtcatattaactggttaaattaatataaatttgtgacatgaccttaactggttaggtaggatatttttcttcatgcaaaaatatgactaataataatttagcacaaaaatatttcccaatactttaattctgtgatagaaaaatgtttaactcagctactataatcccataattttgaaaactatttattagcttttgtgtttgacccttccctagccaaaggcaactatttaaggaccctttaaaactcttgaaactactttagagtcattaagttatttaaccacttttaattactttaaaatgatgtcaattcccttttaactattaatttattttaaggggggaaaggctgctcataattctattgtttttcttggtaaagaactctcagttttcgtttttactacctctgtcacccaagagttggcatctcaacagaggggactttccgagaggccatctggcagttgcttaagatcagaagtgaagtctgccagttcctcccaggcaggtggcccagattacagttgacctgttctggtgtggctaaaaattgtcccatgtggttacaaaccattagaccagggtctgatgaattgctcagaatatttctggacacccaaatacagaccctggcttaaggccctgtccatacagtaggtttagcttggctacaccaaaggaagccatacagaggctaatatcagagtattcttggaagagacaggagaaaatgaaagccagtttctgctcttaccttatgtgcttgtgttcagactcccaaacatcaggagtgtcagataaactggtctgaatctctgtctgaagcatggaactgaaaagaatgtagtttcagggaagaaaggcaatagaaggaagcctgagaatacggatcaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcaggaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgtt ag

Example 1.1 Vector construction

Sequences for ANTIBODY A originated from the 2nd maturation round afterprimary screening of the MorphoSys HuCAL Library, a synthetic phagedisplay library. The DNA for ANTIBODY A was originally provided invectors pMorph from MorphoSys, Germany and corresponds to Fab expressingvectors in FIG. 2 in WO 03/070760, appendix p 6/43. In the vectorconstruction for the purposes of the present invention, vectors pEE6.1and pEE 14.4 (both commercially available from Lonza Biologics areencoded to obtain a construct with both chains in one vector, seeappended FIG. 1; see WO 87/04462 or WO 89/01036. The following cloningstrategy was applied:

The Ig kappa chain was isolated from vector MS-Roche #7.9.H7_Ig_kappachain (as described in WO 03/070760) by PCR with primerACGTAAGCTTGCCGCCACCATGGTGTTGCAG (sense, HindIII; SEQ ID NO. 29) andprimer ACGTGAATTCCTAACACTCTCCCCTGTT (antisense, EcoRI; SEQ ID NO. 30),inserted into pCR 2.1 Topo TA and the insert was completely sequenced.The Ig kappa chain insert was removed from pCR Topo 2.1 by HinDIII/EcoRIdigest and ligated into vector pEE14.4 as HindIII/EcoRI insert.

The Ig gamma 1 heavy chain was cloned from vector pMorph MS-Roche#7.9.H7_IgG1 by PCR with primer ACGTAAGCTTGCCGCCACCATGAAACACCTG (sense,HindIII; SEQ ID NO: 31) and primer ACGTGAATTCTCATTTACCCGGAGACAG(antisense, EcoRI; SEQ ID NO: 32), inserted into pCR 2.1 Topo TA, andthe insert was completely sequenced. The Ig gamma1 heavy chain insertwas removed from pCR Topo 2.1 by HindIII/EcoRI digest and ligated intovector pEE 6.4 as HindIII/EcoRI insert. The heavy chain expressioncassette was removed from pEE 6.4 IgG1 by NotI/SalI digest and theisolated fragment was inserted into SalI/NotI digested pEE14.4 kapparesulting in the final double-gene construct pEE 14.4 mAb-31.

Example 1.2 Transfection of CHO Cells and Expression of ANTIBODY A

Transfections were carried out according to standard protocols. The hostcell line CHO K1 was derived from the Lonza Biologics working cell bank(WCB) # 028-W2 (Lonza, 2002, 1-179) and the host cell line CHO K1 SV wasderived from the Lonza Biologics master cell bank (MCB) #269-M (Lonza,2003, 1-87).

Adherent CHO K1 cells derived from the WCB # 028-W2 were transfectedwith the vector pEE 14.4 MAb31 containing both heavy and kappa lightchain genes by liposomal transfection (Fugene, Roche Diagnostics).Transfection isolates were selected in DMEM, GS supplement (both JRHBiosciences), 10% dialysed FCS (PAA Laboratories, CoS#R0-CEP2001-083-Rev 00) and 50 μM methionine sulphoximine (MSX from Sigma). 2weeks later, colonies were picked and transferred to 96well plates andtested with ELISA for antibody production. 4 colonies with the highestexpression of ANTIBODY A were cloned by serial limited dilution toobtain single-cell derived cultures, of which 82 clones were derivedafter one week and expanded.

One of these clones was selected as the one with the highest specificproduction rate of 48 pg/cell/day in adherent state. It was furthersub-cloned by limiting dilution to obtain the good producers expressingANTIBODY A with high stability (Pu, (1998) Mol Biotechnol, 10, 17-25).Additionally, a suspension variant of the CHO K1 cells, the CHO K1 SVcells from MCB #269-M were transfected with the vector pEE 14.4 MAb31 byelectroporation. Transfectants were selected as before and the resultingclones were subjected to single cell cloning by limiting dilutionresulting in several high producer clones of ANTIBODY A.

Example 1.3 Adaptation of Clones Expressing ANTIBODY A to SuspensionCulture

Best growth properties of CHO K1 clones were determined in DHI mediawith different protein hydrolysates: Cells were finally adapted to DHImedia w/o glutamine (Invitrogen), which is a mixture of DMEM, Ham's F12and IMDM in the respective proportions of 1:1:2 (v:v:v) (Schlaeger andSchumpp, 1992, J Immunol Methods, 146, 111-20) with the followingmodifications: soy and rice hydrolysate: 0.2% soy HyPep 1510 and 0.2%rice HyPep 5603 (Kerry Bioscience), 0.03% Pluronic F68 (Invitrogen), 25μM MSX (Sigma) and 5% dialysed FCS (PAA Laboratories). FCS concentrationwas decreased gradually until cells did grow exponentially in serum-freemedia DHI media. Primary seed banks in serum-free DHI media were frozenfor the several recombinant cell clones

CHO K1 SV clones were adapted from DMEM containing 10% dialysed FCS tosuspension culture in the chemically defined CD-CHO media with 25 μM MSX(Gibco-Invitrogen) in a two step procedure (Lonza). Cell banks werecreated in CD-CHO. Optionally, any other serum-free and protein-freemedia for CHO cells could be used for suspension culture and as a basefor expressing the antibody.

Example 2 Production of ANTIBODY A Production of ANTIBODY A (byFed-Batch Fermentation)

CHO clones were prepared for fermentation from stock cultures in eithershake flasks or spinner cultures as follows:

A cryo vial of the respective clones was thawed in the respectiveculture media containing 25 μM MSX in a 100 ml shake flask or spinnerwith a nominal volume of 50-75 ml.

Cells were then expanded in consecutive splits of 1:5 to reach a stockculture of 400-500 ml volume in either shake flasks or spinners. Cellsused for inoculation of fermenters could be derived from these stockcultures up to 90 days after thawing. The seed train constitutes of a2×1000 ml step in 2 L shake flasks or spinners, followed by inoculationof a 10 L fermenter as a further vessel. Alternatively, the 10 Lfermenter could function as the fed-batch vessel itself or as inoculatefor the 100 L fed-batch fermenter. MSX was present in the culture mediafor selection until the inoculation of the 10 L fermenter where it wasexcluded.

Fermentation Process:

Day 0: Start with 3-4×10⁵/ml cells (1:4-1:5 split from seed culture)Day 2-3: start of feeding, cell density should be above 1.5×10⁶/ml.Feeding: Continuous or bolus feed at 2% per day.

The isoform composition of ANTIBODY A was monitored throughout thefermentation by ion exchange chromatography (see below).

Day 14-18: When the viability of the cells started to drop (50%) and theexpected titers were reached, the cell supernatant was harvested bycentrifugation and/or filtration and filter-sterilised. It was storedaseptically and further processed as described in the next section.

The fermentation was carried out in accordance with standard protocols,see e.g. Werner, (1993), Arzneimittelforschung, 43, 1242-9 or Rendall,(2003). Proceedings of the 18th ESACT meeting, May 11-14, 2003, 1,701-704).

Example 3 Purification of ANTIBODY A

The purification process was based on three chromatographic steps and adiafiltration step: Protein A affinity chromatography, cation exchangechromatography, anion exchange chromatography and diafiltration using a100 kD membrane. The gel types and column sizes were 11 MabSelect (GEHealthcare, Art. 17-5199, column diameter 9 cm, bed length 18+/−2 cm),0.4 l CM-Toyopearl 650M (Toso Bioscience, Art. 007972, small ioncapacity=85 microequivalents/ml, diameter 5.0 cm, bed length 20+/−2 cm),1.3 l Q-Sepharose FF (GE Healthcare, Art. 17-0510-04), diameter 9 cm,bed length 20+/−2 cm. Columns were run at room temperature. Fractionswere stored at 2-8° C. Detection was at 280 nm. A Biomax 100ultrafiltration module with an area of 0.1 m² (Millipore Corp. Art.P2B100A01) was used for concentration and diafiltration.

Protein A Chromatography

The following solutions were prepared using purified water:

Solution A (equilibration buffer): 25 mM Tris, 25 mM NaCl, 5 mM EDTA,adjusted to pH 7.1+/0.1 by HClSolution B (washing buffer 1): 100 mM acetic acid adjusted to pH4.5+/−0.1 by NaOHSolution C (elution buffer): 100 mM acetic acid adjusted to pH 3.2+/−0.1by NaOHSolution D (washing buffer 2): 100 mM acetic acid, 75 mM NaCl, pH3+/−0.1Solution E: (regeneration buffer): 2 M guanidinium hydrochloride, 100 mMTris, adjusted to pH 7.5+/−0.1 by HClSolution F (storage buffer): 200 mM benzyl alcohol, 100 mM acetic acid,adjusted to pH 5.0+/0.1 by sodium hydroxide

The column was first equilibrated with 3 bed volumes of solution A

In the following it was charged with the clarified cell culturesupernatant (45 l, 386 mg/l antibody)

washed with 5 bed volumes of solution A,washed with 3 bed volumes of solution B,eluted with 3.5 bed volumes of solution C and the eluate was collected,washed with 3 column volumes of solution D andregenerated with 2 column volumes of solution Eequilibrated with 3 bed volumes of buffer Aand washed with bed volumes of buffer F for storage.

A linear flow rate of 100 cm/h was used for all chromatographic steps.

The column load was 17.4 g antibody/l Mabselect gel and the yield forthe total mixture of isoforms was 96%.

Viral Inactivation

The following solution was prepared using purified water:

Solution G (adjusting solution): 2 M sodium acetateThe pH of the protein A eluate was adjusted to a pH between 3.5 to 3.7by addition of concentrated acetic acid or 2 M sodium acetate (solutionG). It was stirred for 15 min and than adjusted to pH 4+/−0.1 by adding2 M sodium acetate (solution G).

Cation Exchange Chromatography

The following solutions were prepared using purified water:

Solution H (equilibration buffer): 100 mM acetic acid, adjusted to pH4.0+/−0.1 by NaOHSolution I (elution buffer 1): 250 mM sodium acetate withoutpH-adjusting, pH 7.8-8.5Solution J: (elution buffer 2): 500 mM sodium acetate without pHadjusting, pH 7.8-8.5Solution K (regeneration solution): 0.5 M sodium hydroxideSolution L (storage buffer): 0.01 M sodium hydroxide

The column was first regenerated with 2 bed volumes of solution K andthen equilibrated with 5 bed volumes of solution H.

In the following it was charged with an aliquot of the protein A eluateand washed with 1 bed volume of solution H.

Enclosed it was eluted with 6 bed volumes of solution I. In this step amixture of the double-glycosylated and the mono-glycosylated isoformseluted. In the next step 3 bed volumes of solution J were used to elutethe non-glycosylated isoforms.

After use the column was regenerated with 2 bed volumes of solution K,stored for 24 h in this buffer and was then washed again with 2 bedvolumes of solution K. For storing it was washed with 3 bed volumes ofsolution L.

An example chromatogram is shown in FIG. 3.

Fractions of the chromatography were analyzed by analytical IEX asdescribed below.

A linear flow rate of 100 cm/h was used for all chromatographic steps.

The column load was 14.3 g antibody/l CM-Toyopearl 650 M, and the yieldwas 79% for the mixture of double-glycosylated and mono-glycosylatedisoforms and 6.2% for the non-glycosylated isoforms.

Flow Through Chromatography Using Q-Sepharose FF

The following solutions were prepared in purified water:

Solution M (dilution buffer): 37.5 mM Tris, adjusted to pH 7.9+/−0.1 byacetic acidSolution N (adjusting solution): 2 M TrisSolution O (equilibration buffer): 83 mM sodium acetate, 25 mM Tris, pH7.5+/−0.1Solution P (regeneration buffer 1): 0.5 M NaOH/1M NaClSolution Q (regeneration buffer 2): 0.2 M acetic acid/1 M NaClSolution R (storage buffer): 0.01 M NaOH

The eluate from the CMT column (acidic) was first diluted 1:3 withsolution M and then adjusted to pH 7.5 with solution N.

The column was first equilibrated with 2 bed volumes of solution O andin the following the diluted eluate from the CMT column was processedover the column and the flow through was collected. Product was washedoff the column with solution with solution O until the absorption at 280nm was lower than 0.1 (flow through collected).

The column was regenerated with 1.5 bed volumes of solution P, storedfor 1 h and then regenerated with another 1.5 bed volumes of solution P.Then the column was regenerated with 2 bed volumes of solution Q andwashed with 3 bed volumes of solution R and stored.

A linear flow rate of 100 cm/h was used for all chromatographic steps.

The column load was 3.5 g antibody/l Q Sepharose FF, and the yield was91% for the mixture of double-glycosylated and mono-glycosylatedisoforms.

Diafiltration

The following solution was prepared using purified water:

Solution S (diafiltration buffer): 20 mM Histidine, adjusted to pH 5.5by HCl

A filter holder Pellicon 2 (Millipore Corp.) was equipped with 1ultrafiltration module type Biomax 100 (Millipore Corp., area=0.1 m²,Art.P2B100A01). A WATSON-MARLOW 501 U pump equipped with a siliconetubing was used for pumping. The system was rinsed with buffer O andthen 3.8 litres (1.1 g antibody/l) of the flow through from QSchromatography (adjusted tp pH 5.5 by concentrated acetic acid) wereconcentrated to 250-300 ml within 1 h at 4-11° C. In the following adiafiltration (V=const.) against 3 litres of buffer S (about 10 volumes)was performed (4-11°). Finally the product was sterile filtrated using aMillipac 20 filter (Millipore Corp.). The yield of theultrafiltration/diafiltration step was 91%. The concentration of theproduct was 15 mg/ml. The product could be frozen at −70° C.

Analytical IEX Method for Analysis of Fractions Column: Mono-S HR 5/5(GE Healthcare, Art. 17-0547-01)

Buffer 1: 50 mM Morpholinoethansulfonic acid, adjusted to pH 5.8 bysodium hydroxideBuffer 2: 50 mM Morpholinoethansulfonic acid, 1 M NaCl adjusted to pH5.8 by sodium hydroxideFlow rate: 1 ml/min

Detection: 280 nm

Sample load: 36-72μ

Gradient: Time % Buffer 2

0 min 0 1 0 25 63 27 63 28 0 35 0

An exemplary chromatogram is given in FIG. 2

Yields

Step Isoform Step yield MabSelect (Protein A) Mixture of all isoforms96% CM-Toyopearl 650 M Mixture of mono-glycosylated 79% ANTIBODY A anddouble- glycosylated ANTIBODY A Content of non-glycosylated ANTIBODY A <0.5% Non-glycosylated ANTIBODY A 6.2%  Q-Sepharose FF Mixture ofmono-glycosylated 91% ANTIBODY A and double- glycosylated ANTIBODY AContent of non-glycosylated ANTIBODY A < 0.5% Concentration and Mixtureof mono-glycosylated 91% Diafiltration ANTIBODY A and double-glycosylated ANTIBODY A Content of non-glycosylated ANTIBODY A < 0.5%

Example 4 Characterization of ANTIBODY A Isoforms by SDS-PAGE

SDS-PAGE analysis was carried out using standard protocols with 4-12%NuPage gradient Bis-Tris gel (Invitrogen) and marker MARK12 (Invitrogen)as control. 1-3 ug of ProteinA purified supernatants from fermentations(Prod 01, 02, 03) or spinner cultures (all other lanes) were loaded perwell. The analysis under reducing conditions resulted in a single bandfor peak 1 (double-glycosylated ANTIBODY A), a double band for peak 2(mono-glycosylated ANTIBODY A) and a single band for peak 3(non-glycosylated ANTIBODY A) in the range of the molecular weight ofthe heavy chains. The molecular weights of the two bands of peak 2corresponded to the molecular weights of peak 1 and peak 2,respectively.

Similar results were obtained employing several expression systems like:transient transfection in HEK 293 EBNA cells, transient transfection inCHO cells, and stable expression in CHO cells.

Example 5 Characterization of ANTIBODY A Isoforms by Mass Spectrometric(MS) Analysis

A complete antibody mass profile of all ANTIBODY A isoforms wasdetermined by electron spray ionization mass spectroscopy (ESI-MS).

For this, samples of ANTIBODY A were prepared under non-reducingconditions. The samples were desalted into 2% formic acid and 40%acetonitril by G25 gel filtration and used for ESI-MS analysis in a Q-Tof 2 or LCT-mass spectrometer instrument from Waters.

A separation by molecular mass is obtained with a difference of 1623between non-glycosylated ANTIBODY A and mono-glycosylated ANTIBODY A.The expected mass for non-glycosylated ANTIBODY A from the amino acidsequence is 145,987 Da, which is in good agreement with theexperimentally determined mass of 145,979 Da. Similarly, mono- anddouble-glycosylated ANTIBODY A isoforms differ by 1624 Da as indicatedin FIG. 4. The observed differences in molecular masses are compatiblewith N-glycosylation patterns that are described in more detail hereinbelow.

Example 6 Asn-52 Glycosylation Structure of ANTIBODY A

Asn52 is part of the sequence aaa-aaa-Asn-Ala-Ser-aaa-aaa of thevariable part of the heavy chain, which corresponds to theN-glycosylation consensus sequence Asn-aaa-Ser/Thr. N-linkedglycosylation of Asn52 was confirmed by tryptic peptide mapping ofANTIBODY A isoforms and mass spectrometric evaluation of peptide HC/T4containing Asn52. In tryptic peptide maps of non-glycosylated ANTIBODYA, exclusively a peptide corresponding by mass to non-glycosylated HC/T4peptide appears, indicating that Asn52 was not glycosylated, whereas inthe mono- or double glycosylated ANTIBODY A peptides were detectedcorresponding by mass to HC/T4 containing N-linked sugar structures.

To further confirm glycosylation of the consensus sequence in thetryptic peptide HC/T4 of the heavy chain, the glycosylated HC/T4 peptidewas isolated from peptide maps of glycosylated ANTIBODY A isoforms andanalysed by MALDI-mass spectrometry before and after incubation withN-glycosidase F. Before N-Glycosidase F treatment, masses were obtainedcorresponding to HC/T4 peptide containing N-linked sugar structures.However, the mass of HC/T4 peptide treated with N-Glycosidase Fcorresponded to the mass expected to non-glycosylated HC/T4+1 Da, asexpected if a sugar chain was removed from the asparagine byN-Glycosidase F (Asn to Asp-conversion).

The presence of N-acetyl-neuraminic acids at the sugar structuresattached to Asn52, furthermore, indicates the presence of N-linkedcomplex and hybride type sugar structures. For this, glycosylatedANTIBODY A isoforms were treated with N-Glycosidase F, which removesN-sugar at Asn306, but not at Asn52 and with or without Neuraminidaseand analysed after separation of HC and LC by denaturation and reductionand desalting. The masses obtained for the HC from both approachesdiffered by about 291 Da or 582 Da corresponding to one or 2 sialicacids. From this, it also was concluded that N-linked sugars of thecomplex and/or hybride type were attached to Asn52.

This Asn-52 glycosylation, an N-glycosylation, predominantly consistedof sugar structures of the biantennary complex type (≧75%; mainly80-90%) without core fucosylation and highly sialidated with up to 80%of the complex type antennae containing N-acetyl-neuraminic acids. Minorsugar structures belonged to the biantennary hybrid and the oligomannosetype (≦25%), respectively (FIG. 5 or FIG. 27). Common to all Asn52glycosylation structures was the resistance to cleavage by N-glycosidaseF from intact ANTIBODY A.

Example 7 Asn306 Glycosylation Structure of ANTIBODY A

As pointed out above, ANTIBODY A contained attached to asparagine 306(Asn306) in the Fc-part of the heavy chain (HC) an antibody typeglycosylation consisting of a complex biantennary oligosaccharide chain.It is well known that antibodies contain different isoforms of such acomplex bi-antennenary oligosaccharide chain, varying in the degree ofterminal galactosylation, sialyation and in the degree of corefucosylation. In addition it is known that the degree of lacking corefucosylation in the Fc-located sugar chain is important for in vivoefficacy of antibodies, as it is well accepted that the degree of corefucosylation modulates effector functions of antibodies.

For ANTIBODY A major, antibody typical variations (Routier (1997),Glyoconjugate 14(2), 201-207; Raju (2003), BioProcess International,44-52) in the Fc-located sugar chains attached to Asn306 were foundregarding terminal galactosylation and core fucosylation.

The heterogeneity in the degree of terminal galactosylation (G0:G1:G2structures) was determined to about 35-40% G0-structures, about 45%G1-structures and about 15-20% G2-structures (for schematicdemonstration of the structures see FIG. 6 or FIG. 27).

The content of Fc-sugar structures lacking core fucosylation, i.e.lacking the fucose unit attached to the innermost N-acetyl-glucosamineof the core sugar structure, probably is important for an antibody, asthe presence or absence of this fucose unit may modulate the binding ofthe antibody to Fc-receptors of effector cells, thereby influencingactivity of these cells.

For ANTIBODY A the relative content of sugar chain isoforms lacking corefucosylation at Asn 306 was determined by two different methods, asdescribed in the following:

A) Mass Spectrometry of Complete, Glycosylated HC:

Samples of ANTIBODY A were denatured and reduced into light chain (LC)and glycosylated HC in the presence of 6M guanidine-hydrochloride and250 mM TCEP. The reduced samples were desalted into 2% formic acid and40% acetonitrile and used for ESI-MS analysis in a Q-T of 2 or LCT-massspectrometer instrument from Waters. From the m/z spectra obtained therelative content of the individual oligosaccharide isoforms werecalculated by peak height of glycosylated HCs containing the individualoligosaccharide isoforms from selected single m/z states. Forcalculation of the relative content of sugar structures lacking corefucosylation, peak height of G0-structure lacking core fucose (G0-Fuc)related to the sum of G0+(G0-Fuc).

The respective carbohydrate structures were assigned according to thedifferences of the masses obtained for glycosylated HC and for HC, whoseoligosaccharide structures were removed by incubation with N-GlycosidaseF prior to MS-analysis in control experiments.

B) Chromatographic Analysis of Released Oligosaccharides by HPEAC-PAD:

Samples of ANTIBODY A were incubated with N-Glycosidase F in sodiumphosphate buffer at pH 7.2 in order to release the oligosaccharidechains from Asn306 (the sugar structures at Asn52 were not removed fromthe intact, non-denatured antibody under the conditions used). Thereleased sugar chains were separated from the ANTIBODY A protein bycentrifugation filtration and were analysed on a Carbo Pac PA200 columnfrom Dionex in a BioLC system, using a sodium acetate gradient at strongalkaline pH (pH, 13). The column used was capable of resolvingnon-fucosylated from fucosylated oligosaccharide chains. Assignment ofthe individual peaks obtained to respective carbohydrate structures wasdone by comparing the retention times to the ones of suitableoligosaccharide standards analysed on the Carbo Pac PA200 column and bydetermining the molar mass of the peaks separated and collected by MALDImass spectrometry, respectively. For calculation of the relative contentof sugar structures lacking core fucosylation, the sum of area-% of allstructures lacking core fucose was formed.

The analysis of several batches (combinations of double- andmono-glycosylated ANTIBODY A isoforms) and of purified ANTIBODY Aisoforms, respectively, revealed that the content of non-fucosylatedAsn306 linked oligosaccharide chains was in the range of ˜14%-27%(measured by MS) and 6%-26% (measured by HPAEC-PAD), respectively.

Example 8 Determination of K_(D) Values for ANTIBODY A COMPOSITION andIsoforms (e.g. Non-, Mono- or Double-Glycosylated Antibody of theInvention) Binding to Aβ1-40 and Aβ1-42 Fibers In Vitro by SurfacePlasmon Resonance (SPR)

Binding of ANTIBODY A to fibrillar Aβ was measured online by surfaceplasmon resonance (SPR), and the affinities of the molecularinteractions were determined as follows: Biacore2000 and Biacore3000instruments were used for these measurements. Aβ1-40 and Aβ1-42 fiberswere generated in vitro by incubation of synthetic peptides at aconcentration of 200 μg/ml in 10 mM Na-acetate buffer (pH 4.0) for threedays at 37° C. Electron microscopic analysis confirmed a fibrillarstructure for both peptides, Aβ1-40 showing predominantly shorter (<1micron) and Aβ1-42 predominantly longer (>1 micron) fibers. These fiberswere assumed to represent aggregated Aβ peptides in human AD brain moreclosely than ill-defined mixtures of amorphous aggregates andunstructured precipitates. The fibers were diluted 1:10 and directlycoupled to a CM5 as described in the Instruction Manual of themanufacturer (BIAapplication Handbook, version AB, Biacore AB, Uppsala,1998).

This coupling procedure included an activation step, during which thecarboxylic acid groups on the surface were transferred into chemicallyreactive succinimide ester groups by contacting the surface with anaqueous mixture of N-hydroxysuccinimide and1-ethyl-1-(3-diaminopropyl)-carbodiimide hydrochloride, and animmobilization step, during which the activated surface was contactedwith the fibres dissolved in 10 mM acetate buffer (pH 4.5) at 200-350resonance units (1 resonance unit (RU) corresponds approximately to asurface loading of 1 picogram/mm²). The fiber loaded surface was thencontacted with the antibody solutions in the concentration range 200nM≧C≧0.15 nM. Typical time dependent response curves (=sensograms)monitored during the association phase (during contact with buffer) andthe dissociation phase (subsequent contact with buffer) are shown inFIG. 7.

The K_(D) values for binding to Aβ1-40 and Aβ1-42 fibers of the ANTIBODYA isoforms are given in the table below. Briefly, K_(D) values werecalculated by Scatchard type analysis using concentration dependentequilibrium binding responses. These equilibrium binding constants couldbe obtained in two ways.

Due to the very slow association process at low antibody concentrationcontact intervals to reach equilibrium were very long (FIG. 7).Nevertheless, such contact intervals could be realized on Biacoreinstruments and the experimental equilibrium responses could besubjected to a Scatchard analysis.

Equilibrium binding data were also obtained by extrapolating shortertime dependent association curves to infinity. These theoreticallyobtained equilibrium binding levels were then again used for thedetermination of the K_(D) values.

Independently from the way of determining equilibrium sensor responsescurvilinear Scatchard plots were obtained. From the curvilinearScatchard plot a higher (bivalent) and lower (monovalent) affinityinteraction was derived for ANTIBODY A isoforms derived from the secondaffinity maturation cycle. These two affinities represent the lower andupper K_(D) values of the range indicated the following table:

1-40 1-40 high affinity K_(D) low affinity K_(D) values (nM) values (nM)ANTIBODY A extrapolation 0.49 27.25 COMPOSITION stdev 0.10 8.40 (mixtureof mono- and equilibrium 0.41 21.00 double-glycosylated ANTIBODY A)double-glycosylated extrapolation 1.43 18.51 ANTIBODY A stdev 0.02 22.33equilibrium 1.54 29.00 mono-glycosylated extrapolation 0.25 7.55ANTIBODY A stdev 0.02 2.06 equilibrium 0.12 11.10 non-glycosylatedextrapolation 0.19 1.99 ANTIBODY A stdev 0.03 0.15 equilibrium 0.42 2.82

The table above shows K_(D) values of the low affinity complex(monovalent) and the high affinity complex (bivalent) formed by theinteraction of ANTIBODY A isoforms and Aβ1-40 fibrils as determined bysurface plasmon resonance. K_(D) determined by using extrapolatedequilibrium responses (marked “extrapolation”) and KD values determinedby using experimentally determined equilibrium responses (marked“experimental”) are given. The extrapolated values were determined atleast six times and a standard deviation is given. The K_(D)'s based onexperimental and extrapolated equilibrium sensor responses are equalwithin the limits given by these standard deviations.

Example 9 Epitope Mapping of ANTIBODY A COMPOSITION and Isoforms (e.g.Non-, Mono- or Double-Glycosylated Antibody of the Invention) by PepspotAnalysis with Decapeptides

An epitope (antigenic determinant) can be linear or conformational. Theherein described dual epitope specificity was defined by reactivity ofthe antibody with two non-sequential linear peptides.

Epitope mapping approaches which were used to define specific epitoperecognition are based on ELISA technology with hexapeptide conjugatescoated on to microplates or on pepspot technology. The latter technologyallows the detection and quantitation of the antibody by protocols thatare commonly known for Western Blotting of proteins to PVDF membranes.

Applied epitope mapping technologies are designed to specifically detectlinear epitopes, whereas they cannot be applied to map more spatiallycomplex epitopes like discontinuous or conformational epitopes.Techniques available for conformational or discontinuous epitopemapping, like domain scanning and combinatorial peptide arrays requirelong peptides up to 36 amino acids (domains) or combined peptides eachconsisting of 12 amino acids.

The applied techniques are therefore considered to be specific forlinear epitopes, excluding that conformational epitopes eitherdiscontinuous or discontinuously scattered epitopes are involved.

In conclusion, the presented data show that the two regions within theAβ peptide defined herein resemble independent linear epitopessimultaneously recognized based on the unique dual epitope specificityof the investigated antibodies on single hexameric or decamericAβ-peptides.

The following amino acid sequence encompassing Aβ (1-42) was dividedinto 43 overlapping decapeptides with a frameshift of 1 amino acid. Thenumbers refer to the essential amino acids from the Aβ1-40 sequencewhich have to be present in the decapeptide for optimal binding ofantibody.

ISEVKM¹DAEF RHDSGYEVHH QKLVFFAEDV GSNKGAIIGL MVGGVVI⁴²ATV IV (SEQ ID NO:4). Accordingly, DAEF RHDSGYEVHH QKLVFFAEDV GSNKGAIIGL MVGGVVIA (SEQ IDNO: 3) represents amino acids 1 to 42 of Aβ4/β-A4 peptide.

The 43 decapeptides were synthesized with N-terminal acetylation andC-terminal covalent attachment to a cellulose sheet (“pepspot”) by acommercial supplier (Jerini BioTools, Berlin). The cellulose sheet wasincubated for 2 hours on a rocking platform with monoclonal antibody (1μg/ml) in blocking buffer (50 mM Tris.HCl, 140 mM NaCl, 5 mM NaEDTA,0.05% NP40 (Fluka), 0.25% gelatin (Sigma), 1% bovine serum albuminfraction V (Sigma), pH 7.4). The sheet was washed 3 times 3 minutes on arocking platform with TBS (10 mM Tris.HCl, 150 mM NaCl, pH 7.5). It wasthen pressed on filter paper, wetted with cathode buffer (25 mM Trisbase, 40 mM 6-aminohexane acid, 0.01% SDS, 20% methanol) and transferredto a semi-dry blotting stack with the peptide side facing a PVDFmembrane (Biorad) of equal size.

The semi-dry blotting stack consisted of freshly wetted filter papers(Whatman No. 3) slightly larger than the peptide sheet:

3 papers wetted with Cathode bufferthe peptide sheeta sheet of PVDF membrane wetted with methanol3 papers wetted with Anode buffer 1 (30 mM Tris base, 20% methanol)3 papers wetted with Anode buffer 2 (0.3 mM Tris base, 20% methanol)

The transfer was conducted at a current density between Cathode andAnode of 0.8 mA/cm² for one hour which was sufficient to elute theantibody completely from the cellulose sheet and transfer it on the PVDFmembrane. The PVDF membrane was immersed in blocking buffer for 10minutes. Goat anti-human IgG(H+L) labeled with fluorochrome IRdye800(Rockland code# 609-132-123) was added at 1:10'000 dilution in Odysseyblocking buffer (Li-Cor) and further diluted 1:1 with PBS, 0.05%Tween20. The membrane was incubated on a rocking platform for 1 hour. Itwas washed 3×10 minutes with TBST (TBS with 0.005% Tween20). Themembrane was dried and scanned for 800 nm fluorescence using a longwavelength fluorescence scanner (Odyssey) as shown in FIG. 8.

The exact assignment of antibody-reactive spots was achieved by markingthe PVDF membrane with a needle puncture. The epitopes of the antibodyin question was defined as the minimal amino acid sequence in reactivepeptides. The fluorescence intensity was integrated over each spot andrecorded as relative fluorescence unit (RFU). For comparison two mousemonoclonal antibodies (BAP-1 which is equivalent to antibody 6E10 (Kim(1998).) with specificity for the N-terminal domain, and BAP-44 which isequivalent to antibody 4G8 (Kim (1998),) with specificity for the middledomain) were analyzed in the same way, except for using anti-mouse Iginstead of anti-human Ig for detection.

It is of note that affinity maturation and conversion of the monovalentFab fragments into full-length IgG1 antibodies results usually in somebroadening of the epitope recognition sequence as indicated by pepspotand ELISA analyses. This may be related to the recruitment of morecontact points in the antibody-antigen interaction area as a consequenceof the affinity maturation or to a stronger binding to the minimalepitope such that also weak interactions with adjacent amino acids canbe detected. The latter may be the case when Aβ-derived peptides areprobed with full-length IgG antibodies. As illustrated in the tablebelow, the recognition sequences of the N-terminal and middle epitopeswere extended by up to three amino acids when parent Fabs andcorresponding fully maturated IgG antibodies were compared. However, ithas to be kept in mind that the decapeptides were modified for covalentattachment at the C-terminal amino acid and this amino acid maytherefore not easily be accessible to the full-length antibody due tosteric hindrance. If this is the case the last C-terminal amino aciddoes not significantly contribute to the epitope recognition sequenceand a potential reduction of the minimal recognition sequence by oneamino acid at the C-terminal end has to be considered in the pepspotanalysis as used in the present invention.

Amino acid Amino acid antibody position position double-glycosylatedANTIBODY A 3-4 (1-10) 18-24 (17-26) mono-glycosylated ANTIBODY A 4-5(3-11) 20-26 non-glycosylated ANTIBODY A 3-4 20-24 ANTIBODY ACOMPOSITION 3-5 (3-11) 19-26 (Mixture of mono- and double- glycosylatedANTIBODY A isoforms (1:1)) BAP-44 (mouse monoclonal) 19-21 BAP-1 (mousemonoclonal) 4-6

The table above relates to a pepspot analysis of binding full-length IgGantibodies to decapeptides on a cellulose sheet. The numbers refer tothe amino acid position in the Aβ1-40 sequence which have to be presentin the decapeptide for binding of antibody. A further extension to theepitope is indicated in brackets in order to indicate the flanking aminoacids that are required to achieve maximum binding.

Example 10 De-Polymerization Assay Employing ANTIBODY A Isoforms (e.g.Non-, Mono- or Double-Glycosylated Antibody of the Invention) whichInduces Release of Biotinylated Aβ from Aggregated Aβ

The experimental setup to test the potential of ANTIBODY A isoforms toinduce dissociation of aggregated Aβ was as follows:

Biotinylated Aβ1-40 was first incorporated into preformed Aβ1-40/Aβ1-42fibers before treatment with ANTIBODY A isoforms. Liberation ofbiotinylated Aβ was measured using an assay employing a streptavidin-PODconjugate as described below.

Synthetic Aβ when incubated in aqueous buffer over several daysspontaneously aggregates and forms fibrillar structures which aresimilar to those seen in amyloid deposits in the brains of Alzheimer'sDisease patients. The following in vitro assay is suitable to measureincorporation or liberation of biotinylated Aβ into preformed Aβaggregates in order to analyze the Aβ-neutralizing potential of anti-Aβantibodies and other Aβ-binding proteins such as albumin (Bohrmann(1999) J. Biol. Chem. 274, 15990-15995). ANTIBODY A isoforms inducedde-polymerization of aggregated Aβ as measured by the release ofincorporated biotinylated Aβ1-40.

Experimental Procedure:

NUNC Maxisorb microtiter plates (MTP) were coated with a 1:1 mixture ofAβ1-40 and Aβ1-42 (2 μM each, 100 μl per well) at 37° C. for three days.Under these conditions highly aggregated, fibrillar Aβ was adsorbed andimmobilized on the surface of the well. The coating solution was thenremoved and the plates were dried at room temperature for 2-4 hours. Thedried plates could be stored at −20° C. For incorporation ofbiotinylated Aβ the coated plates were incubated with 200 μl/well 20 nMbiotinylated Aβ1-40 in TBS containing 0.05% NaN₃ at 37° C. overnight.After washing the plate with 3×300 μl/well T-PBS, antibodies seriallydiluted in TBS containing 0.05% NaN₃ were added and incubated at 37° C.for 3 hours. The plate was washed and analyzed for the presence ofbiotinylated Aβ1-40. After washing 3× with 300 μl T-PBS astreptavidin-POD conjugate (Roche Molecular Biochemicals), diluted1:1000 in T-PBS containing 1% BSA, was added (100 μl/well) and incubatedat room temperature for 2 hours. The wells were washed 3× with T-PBS and100 μl/well of a freshly prepared tetramethyl-benzidine (TMB) solutionwere added. [Preparation of the TMB solution: 10 ml 30 mM citric acid pH4.1 (adjusted with KOH)+0.5 ml TMB (12 mg TMB in 1 ml acetone+9 mlmethanol)+0.01 ml 35% H₂O₂]. The reaction was stopped by adding 100μl/well 1 N H₂SO₄ and absorbance was read at 450 nm in a microtiterplate reader.

As documented in appended FIG. 9, ANTIBODY A isoforms induceddissociation of aggregated Aβ as measured by the release of incorporatedbiotinylated Aβ1-40. ANTIBODY A isoforms and the mouse monoclonalantibody BAP-1 were similarly active (FIG. 9), whereas the BAP-2, BAP-17and 4G8 antibodies were clearly less efficient in liberatingbiotinylated Aβ from the bulk of immobilized Aβ (data not shown). BAP-1could clearly be differentiated from the glycosylated ANTIBODY Aisoforms by its reactivity with cell surface full-length APP. Antibodieslike BAP-1 with such properties are not useful for therapeuticapplications as potential autoimmunological reactions may be induced. Itis interesting to note that BAP-2, despite its specificity for aminoacid residue 4-6 which was exposed in aggregated Aβ has a clearly loweractivity in this assay indicating that not all N-terminus specificantibodies a priori are equally efficient in releasing Aβ from preformedaggregates. The relatively low efficiency of BAP-17(C-terminus-specific) and 4G8 (amino acid residues 16-24-specific) inthis assay was due to the cryptic nature of these two epitopes inaggregated Aβ. BSA at the concentrations used here had no effect onaggregated Aβ.

The mono-glycosylated isoform exerted higher capacity compared to thedouble-glycosylated isoform to depolymerize aggregated Aβ peptide invitro that may be relevant also in vivo.

Example 11 ANTIBODY A COMPOSITION and Comprising Isoforms (Mono- andDouble-Glycosylated Antibody of the Invention) Capture Soluble Aβ fromHuman Cerebrospinal Fluid (CSF)

The capacity to capture soluble Aβ from human CSF samples was determinedby immunoprecipitation (IP) and semi-quantitative Western blot (WB)analysis. Experimental procedure:

Immunoprecipitation of human CFS samples was done according to thefollowing scheme:

70 ul human CSF 20 ul Incubation buffer (50 mM Tris, 140 mM NaCl, 5 mMEDTA, 0.05% NP-40, 1% BSA, 0.25% Gelatin, 0.25% milk powder, pH 7.2 10ul ANTIBODY A isoforms from stock solutions (1000-10 ug/ml) 100 ul 

The solution was kept for one hour at 4° C. 40 ul protein G Sepharosebeads (Amersham Biosciences #17-0618-01; washed with PBS, 50% slurry)were added and incubated for 2 hours at 4° C. on rotator. Aftercentrifugation at 500 g for 3 minutes at 4° C. the supernatant wasremoved and 200 ul PBS were added to the beads, transferred to Milliporefilter tubes 0.45 um (Millipore #UFC3OHVNB) and centrifuged at 500 g for3 minutes at 4° C. Additional 200 ul PBS was added to beads, vortexedand centrifuged at 2000 g for 3 minutes at 4° C. 45 ul sample buffer1×NuPage with DTT was added and kept for 10 minutes at 70° C. followedby centrifugation at 2000 g for 3 minutes at 4° C.

For SDS-PAGE, 18 ul protein G eluate was applied to NuPage gel 10%Bis-Tris gel together with Aβ₁₋₄₂ (Bachem) as internal standard directlyin sample buffer as standard and run in MES buffer system.

The gel was transferred to Hybond C extra membrane (semi-dry systemNovex) dry membrane 3′ at room temperature. The membrane was transferredinto pre-heated PBS and heated in microwave for 3 min. at 600 W.Blocking was done for 1 hour with SuperBlock Solution (Pierce) andadditional blocking for 1 hour with 5% Milk Powder (Bio Rad) in T-PBS(0.1% Tween20 in PBS).

Incubation was done over night with anti Aβ antibody W02 Antibody(1:1500-1:2000 from The Genetics, Inc. Zürich, Switzerland) at 4° C. ona rotator, followed by washing 3× with T-PBS for 5 min and incubationfor 2 hours at RT with anti-mouse IgG-HRP (Dako) 1:5000 in T-PBS.Another washing 3× with T-PBS for 5 min was followed by incubation withLumiLight Plus for 5 minutes at RT. Western blots were digitized andanalysed by densometry with an Alpha Innotech Digital Camera System.

As documented in FIG. 10, ANTIBODY A COMPOSITION (comprising mono- anddouble-glycosylated ANTIBODY A isoforms) bound efficiently to soluble Aβin human CSF as demonstrated by immunoprecipitation and Western blottingexperiments. Notably, in this assay, mono-glycosylated ANTIBODY A wasmore efficient in capturing soluble Aβ than double-glycosylated ANTIBODYA (FIG. 10).

Example 12 In Vitro Immunostaining of Human Amyloid Plaques by ANTIBODYA COMPOSITION and Isoforms (e.g. Non-, Mono- or Double-GlycosylatedAntibody of the Invention)

Glycosylated ANTIBODY A isoforms were tested for the ability to staingenuine human β-amyloid plaques obtained from brain sections of patientswith severe Alzheimer's Disease by immunohistochemistry analysis usingindirect immunofluorescence. Specific and sensitive staining of genuinehuman β-amyloid plaques was demonstrated.

Cryostat sections of unfixed tissue from the temporal cortex obtainedpostmortem from a patient that was positively diagnosed for Alzheimer'sdisease were labeled by indirect immunofluorescence. A successivetwo-step incubation was used to detect bound ANTIBODY A isoforms, whichwere revealed by affinity-purified goat anti-human (GAH) IgG (H+L)conjugated to Cy3 (# 109-165-003, lot 49353, Jackson Immuno Research).Controls included unrelated human IgG1 antibodies (Sigma) and thesecondary antibody alone, which all gave negative results.

All types of β-amyloid plaques were sensitively and specificallydetected and consistently revealed at an ANTIBODY A concentration of 10ng/ml (FIG. 11).

Specific and sensitive staining of genuine human amyloid-β plaques isdemonstrated for the glycosylated ANTIBODY A isoforms at a concentrationup to 1 μg/ml.

At a concentration of 10 μg/ml a background staining was observed, mostprominent with the non-glycosylated ANTIBODY A isoform. Thenon-glycosylated isoform exerted considerable unspecific stickinessobserved at the surface of glass slides and almost all tissue componentsthat were exposed after the sectioning process in vitro. This appearedto be due to unspecific binding involving ionic and/or hydrophobicinteractions.

Example 13 In Vivo β-Amyloid Plaque Decoration by ANTIBODY A in a MouseModel of Alzheimer's Disease

Glycosylated ANTIBODY A isoforms were tested in a single dose study inPS2APP double transgenic mice (Richards (2003), J. Neuroscience, 23,8989-9003) for their ability to immuno-decorate β-amyloid plaques invivo. The glycosylated ANTIBODY A isoforms were administered i.e. at adose of 1 mg/mouse and after 3 days animals were perfused withphosphate-buffered saline and the brains frozen on dry ice and preparedfor cryosectioning.

Both glycosylated isoforms showed improved and highly effective brainpenetration in vivo (as compared to the non-glycosylated form).Effective brain penetration and specific binding to amyloid-β plaqueswas demonstrated in PS2APP mice, a mouse model for Aβ-relatedamyloidosis.

The presence of the antibodies bound to β-amyloid plaques was assessedusing unfixed cryostat sections either by single-labeled indirectimmunofluorescence with goat anti-human IgG (H+L) conjugated to Cy3(#109-165-003, Jackson Immuno Research) shown in FIG. 12 or followed bycounterstaining with BAP-2-Alexa488 immunoconjugate to visualize theposition and distribution of all β-amyloid plaques types present in thetissue.

An immuno-fluorescence staining approach was used to detect boundANTIBODY A. After adhesion to precooled glass slides, sections werehydrated in PBS and treated with 100% acetone precooled at −20° C. for 2min. Washing with PBS was done twice for two minutes. Blocking ofunspecific binding sites was done either with PBS containing 1% BSA orby sequential incubation in Ultra V block (LabVision) for 5 min followedby a PBS wash and incubation in power block solution (BioGenex) with 10%normal sheep serum for 20 min. After washing with PBS with 10% normalsheep serum slides were incubated with affinity-purified goat anti-human(GAH) IgG (H+L) conjugated to Cy3 (# 109-165-003, lot 49353, JacksonImmuno Research) at a concentration of 15 μg/ml for 1 hour at roomtemperature. A counterstaining for amyloid plaques was by incubationwith BAP-2, a mouse monoclonal antibody against Ab conjugated to Alexa488 at a concentration of 0.5 μg/ml for 1 hour at room temperature.Autofluorescence of lipofuscin was quenched by incubation in 4 mM CuSO4in 50 mM ammonium acetate. After rinsing the slides in bidistilled waterand washing with 2×500 μl/slide PBS, slides were embedded withfluorescence mounting medium (S3023 Dako).

Imaging was done by confocal laser microscopy and image processing forquantitative analysis of colocalizations by IMARIS and COLOCALIZATIONsoftware (Bitplane, Switzerland).

After a single dose of 1 mg per mouse glycosylated ANTIBODY A isoformswere found to penetrate across the blood brain barrier and toeffectively immuno-decorate/bind all β-amyloid plaques after three daysin vivo. Representative images are shown in FIG. 12. This is in clearcontrast to the non-glycosylated form which is not detectable at amyloidplaques.

Example 14 Investigation of Binding of ANTIBODY A Isoforms to AmyloidPrecursor Protein (APP) Expressed on the Surface of HEK293 Cells

The method of flow cytometry is well known in the art. Relative units offluorescence (e.g. FL1-H) measured by flow cytometry indicated cellsurface binding of the respective antibody. A fluorescence shift on APPtransfected HEK293 compared to untransfected HEK293 cells indicated theunwanted reaction with cell surface APP. As an example, antibodies BAP-1and BAP-2 against the N-terminal domain showed a significant shift ofFL-1 signal in HEK293/APP (FIG. 13, thick line, right hand panel)compared to untransfected HEK293 cells (FIG. 13, dotted line, right handpanel). Similarly, BAP-44 antibody (specific for the middle A-betaepitope) showed a similar size shift. In contrast, all ANTIBODY Aisoforms (FIG. 13 left hand panel) (specific for N-terminal and middleA-beta epitopes) showed no significant shift in fluorescence. Theuntransfected HEK293 cells had a higher basal fluorescence than the APPtransfected cells due to different cell size and surface properties. AFACScan instrument was used in combination with the Cellquest ProSoftware package (both Becton Dickinson).

ANTIBODY A isoforms were devoid of reactivity towards cell surface APP(FIG. 13).

Example 15 Morphometrical Analysis of Aβ Plaque Deposition in a MouseModel of Alzheimer's Disease

The capability of ANTIBODY A COMPOSITION or the ANTIBODY A isoforms tolower amyloidosis in vivo was studied in various brain regions(thalamus, neocortex, hippocampus and subiculum) using quantitativecomputer-assisted image analysis of brains of PS2APP mice that receiveda five-month treatment with ANTIBODY A COMPOSITION or ANTIBODY Aisoforms.

Therefore, PS2APP transgenic male mice were injected i.v. with ANTIBODYA COMPOSITION or ANTIBODY A isoforms and vehicle. Seventy-five5-6-month-old PS2APP mice were divided into five groups (A-E),consisting of 15 mice each. Beginning on day 0, each mouse receivedeither 0.1 mL of vehicle (0 mg/kg), or ANTIBODY A preparations (20mg/kg) by bolus i.v. injection via the tail vein. Groups A, B, C, D andE of PS2APP mice received vehicle (histidine-buffered saline), ANTIBODYA COMPOSITION which comprises mono-glycosylated ANTIBODY A anddouble-glycosylated ANTIBODY A and is devoid of non-glycosylatedANTIBODY A as defined above, double-glycosylated ANTIBODY A,mono-glycosylated ANTIBODY A and non-glycosylated ANTIBODY A,respectively.

Immunotolerance against the administered human anti-Aβ antibodies wasinduced by injecting anti-CD-4 antibody (hybridome clone GK 1.5 ascommercially available from ATCC). Monitoring of anti-drug antibodiesindicated that antibody treated animals only develop a moderateimmune-response after more than 16 weeks of treatment and that thedetectable antibodies are either of low affinity or are produced only inlow amounts (data not shown).

After 5 month treatment mice were sacrificed. Unfixed brains weresectioned sagitally, including thalamus, hippocampal formation andcortical areas. From each brain hemisphere 50 sections were prepared isfollows: Starting at lateral level ˜1.92, 5 consecutive series of 5×10μm and 5×20 μm sections were obtained. There was no gap betweenconsecutive sections, resulting in a total tissue usage of 750 μm. Thesection series therefore ends approximately on lateral level 1.20(Paxinos and Franklin, 2003). For quantitative morphometrical analysisevery 10th section was used.

Sections were stained for amyloid deposits with the double-glycosylatedANTIBODY A isoform at a concentration of 5 μg/ml. Stainings against Aβusing a mouse monoclonal antibody (BAP-2) conjugated to Alexa-488fluorophore at 5 μg/ml showed comparable results although withsignificant intracellular and background staining of neurons whichinterfered with the image processing routine described below. Fordetection an affinity-purified goat anti-human (GAH) IgG (H+L)conjugated to Cy3 (# 109-165-003, lot 49353, Jackson Immuno Research) ata concentration of 15 μg/ml for 1 hour at room temperature was applied.After washing with 2×500 μl PBS/slide, slides were embedded withfluorescence mounting medium (S3023, Dako).

Images were acquired using a GenePix Personal 4100A microarray scanner(Axon Instruments, now Molecular Devices, CA, USA). Amyloid-β plaqueload and number was measured using two parameters, namely percentage ofarea covered by amyloid-β plaques and number of amyloid-β plaques usingan unbiased morphometrical method by means of computer assisted imageanalysis. Quantification of plaque load and number was done with MCID M7Elite software (Imaging Research Inc., St. Catherines/Ontario, Canada).The scanned images were enhanced by a detail extractor filter followedby a target accent filter. The resulting image was then binarized,adjusting the threshold according to the staining intensity. Artefacts,blood vessels and edge effects were identified on the original referenceimage and then removed from the binarized image. Regions of interestwere outlined on the reference image. For final quantification, the areaof these regions and the area occupied by plaques as well as the plaquenumber were then measured in the binarized image. Single pixels wereignored. Calculations were made with common spreadsheet software(Microsoft Excel, Redmond/Wash., U.S.A.). The size of plaques wasseparated into 11 groups ranging from <100 to >1000 μm2. Statisticalevaluation was done using a twotailed, heteroscedastic t-test.

For comparison and statistical evaluation, the baseline of amyloidosis(amyloid-β plaque pathology) was determined at study begin with a cohort(15 animals) of non-treated 6 month old PS2APP mice. Results aredepicted in FIGS. 15 to 18 with levels of significance (*: p≦0.05; **:p≦0.01; ***: p≦0.001).

Amyloid plaque reduction was most pronounced in the thalamus region(FIG. 15). The mean reduction of total amyloid-β plaque surface wasdetermined for the antibody treated groups: 64% for ANTIBODY ACOMPOSITION, 70% for double-glycosylated ANTIBODY A, 81% formono-glycosylated ANTIBODY A and 44% for non-glycosylated ANTIBODY A.The mean reduction in total amyloid-β plaque number was found to be 70%for ANTIBODY A COMPOSITION, 78% for double-glycosylated ANTIBODY A, 82%for mono-glycosylated ANTIBODY A and 36% for non-glycosylated ANTIBODYA. Note that significance for non-glycosylated ANTIBODY A was low andobserved variations were considerable.

Amyloid plaque reduction in the neocortical region together with thecorpus callosum is depicted in FIG. 16. The mean reduction of totalamyloid-β plaque surface was determined for the antibody treated groups:19% for ANTIBODY A COMPOSITION, 27% for double-glycosylated ANTIBODY A,30% for mono-glycosylated ANTIBODY A and 10% for non-glycosylatedANTIBODY A. The mean reduction in total amyloid-β plaque number wasfound to be 40% for ANTIBODY A COMPOSITION, 46% for double-glycosylatedANTIBODY A, 42% for mono-glycosylated ANTIBODY A and 11% fornon-glycosylated ANTIBODY A.

Amyloid plaque reduction in the entire hippocampal region is depicted inFIG. 17. The mean reduction of total amyloid-β plaque surface wasdetermined for the antibody treated groups: 12% for ANTIBODY ACOMPOSITION, 24% for double-glycosylated ANTIBODY A, 24% formono-glycosylated ANTIBODY A and 6% for non-glycosylated ANTIBODY A. Themean reduction in total amyloid-β plaque number was found to be 36% forANTIBODY A COMPOSITION, 46% for double-glycosylated ANTIBODY A, 37% formono-glycosylated ANTIBODY A and 3% for non-glycosylated ANTIBODY A.

Amyloid plaque reduction in the subiculum, a high susceptibility regionfor amyloidosis is shown in FIG. 18. The mean reduction of totalamyloid-β plaque surface was determined for the antibody treated groups:2% for ANTIBODY A COMPOSITION, 12% for double-glycosylated ANTIBODY A,5% for mono-glycosylated ANTIBODY A and 1% for non-glycosylated ANTIBODYA. The mean reduction in total amyloid-β plaque number was found to be22% for ANTIBODY A COMPOSITION, 36% for double-glycosylated ANTIBODY A,13% for mono-glycosylated ANTIBODY A and 1% for non-glycosylatedANTIBODY A. ANTIBODY A COMPOSITION and the main N-glycosylation isoforms(double-glycosylated ANTIBODY A and mono-glycosylated ANTIBODY A) showeda comparable effectivity to decrease amyloid-β plaque load and plaquenumber. Reduction of plaque load was most pronounced and statisticallysignificant in regions with low or moderate amyloidosis.

Overall, reduction of amyloid-β plaque number was found statisticallysignificant after treatment with ANTIBODY A COMPOSITION and bothcomprising Asn52 glycosylated isoforms of ANTIBODY A in all measuredbrain regions. In contrast thereto, only a minor effect on amyloid-βplaque number was found in the thalamus and no significant effects onamyloid-β plaque number in the other investigated brain regions wasfound after treatment with the non-glycosylated isoform of Antibody A,which is excluded from ANTIBODY A COMPOSITION after the purification asdetailed in the invention.

We also investigated potency of plaque clearance in relation to theplaque size. Generally, effectivity of tested human anti-Aβ antibodieswas found most pronounced for the clearance of small amyloid-β plaques.This was observed in all brain regions (FIGS. 15 C, 16 C, 17 C and 18C). In contrast, there was only a minimal or non-significant trendobserved for the non-glycosylated isoform of ANTIBODY A.

Comparative analysis of ANTIBODY A and the major Asn52 glycosylationisoforms demonstrate a comparable capacity to lower plaque load, whilethe non-glycosylated isoform has no significant effect on plaquelowering.

Example 16 Pharmacokinetics of In Vivo Binding of ANTIBODY A COMPOSITIONto Amyloid-β Plaques

Two dosing frequencies were compared in order to investigate the bindingkinetics of ANTIBODY A COMPOSITION which comprises mono-glycosylatedANTIBODY A and double-glycosylated ANTIBODY A and is devoid ofnon-glycosylated ANTIBODY A as defined above.

Therefore, PS2APP transgenic male mice were injected i.v. with ANTIBODYA COMPOSITION via the tail vein either 4 times at biweekly intervals at0.05, 0.1 and 0.3 mg/kg or 3 times at monthly interval with 0.075, 0.15and 0.45 mg/kg. For comparison, 0.1 mg/kg was administered once andtwice at biweekly intervals and 0.15 mg/kg twice at monthly interval.Following administration all mice were sacrificed two weeks after lastdosing. Unfixed PS2APP brain tissue was prepared for sagital sectioningbetween lateral ˜1.92 and 1.2 mm according to Paxinos and Franklin,including thalamus, hippocampal formation and cortical areas. Brainswere sectioned at 40 μm using a cryostat.

An immuno-fluorescence ex vivo immuno-staining approach was used todetect bound ANTIBODY A COMPOSITION antibodies. Therefore, brains weresectioned and incubated with the detection antibody, anaffinity-purified goat anti-human (GAH) IgG (H+L) conjugated to Cy3 (#109-165-003, lot 49353, Jackson Immuno Research) at a concentration of15 μg/ml for 1 hour at room temperature. A counterstaining for amyloid-βplaques was done by incubation with BAP-2, a mouse monoclonal antibodyagainst Aβ conjugated to Alexa488 fluorophore at a concentration of 0.5μg/ml for 1 hour at room temperature.

Images were recorded in the occipital cortex close to the cerebellumusing a Leica TCS SP2 AOBS confocal laser scanning microscope asdescribed above. Computer-assisted image processing was performed usingthe IMARIS software (Bitplane, Switzerland). Images of plaques werefirst selected using the crop function of the software for the lowerdoses except the two highest doses of 0.3 and 0.45 mg/kg which requireda different gain setting for linear signal recording. The SURPASSfunction was used to select the positive voxels after thresholding (T)as readout for bound GAH-Cy3 at the site of amyloid-β plaques. Thresholdsettings were 19 and 12 for lower and higher dose groups, respectively.As a control for amyloid-β plaque specificity, images of the GAH-Cy3stainings were compared after double labeling with images of plaquesstained by mouse monoclonal BAP2 conjugated to Alexa488 and recorded ina different channel.

Descriptive statistics to quantitative description of all images wasdone with the IMARIS MeasurementPro software module. Mean voxelfluorescence intensity (MVI) values were determined from selectedamyloid-β plaques in the low dose groups or total signal from images inthe high dose groups. The baseline MVI (B) is due to instrumental noise,tissue scatter signal and autofluorescence of lipofuscin. For backgroundcorrection, B was determined by measuring the average signal intensityat areas apart from amyloid-β plaques and subsequently subtracted fromthe MVI of all measured images (MVI−B=S). Signal intensity (S) valuesresembling averaged intensities on plaques obtained from 3 to 4 imagesfrom each one brain section per mouse and dose group. For comparability,signal intensities were normalized to a reference sample obtained from aprevious study. As reference we used PS2APP mouse brain sections after asingle (lose administration of 0.25 mg/kg. Measurement endpoint was oneweek after dosing

All measured intensity values were normalized to the average intensityat amyloid-β plaques obtained after a single dose administration ofANTIBODY A COMPOSITION at 0.25 mg/kg that was measured one week afterdosing (see following table). The normalized values for mean relativefluorescence intensity of immunopositive amyloid-β plaques were obtainedby CLSM after immunostaining and measuring signal intensities averagedfrom 3 animals per dose group. Plaques without detectable ANTIBODY ACOMPOSITION derived ANTIBODY A were observed only in the lower dosegroups, most likely due to limited or partial occupancy of ANTIBODY ACOMPOSITION derived ANTIBODY A at the plaque surface, which might havebeen lost during the sectioning process. Therefore, only immunopositiveplaques were included for the comparative analysis.

Mean relative fluorescence intensity per dose group after multiple i.v.(BOLUS) administration of ANTIBODY A COMPOSITION in PS2APP transgenicmice is shown in the following table:

Normalized Average Mean Fluorescence Percentage of Intensity ofimmunopositive ANTIBODY A ANTIBODY A COMPOSITION COMPOSITIONimmunonegative Dose - Amyloid-β Plaques amyloid-β plaquesinterval/injections % SD % 0.25 mg - single¹ 100 6 0 0.05 mg -biweekly/4x 53 2 58 0.075 mg - monthly/3x 57 6 39 0.15 mg - monthly/2x106 6 19 0.1 mg - biweekly/1x 59 8 45 0.1 mg - biweekly/2x 83 26 21 0.1mg - biweekly/4x 88 12 2 0.15 mg - monthly/3x 93 19 1 0.3 mg -biweekly/4x 148 24 0 0.45 mg - monthly/3x 184 20 0 ¹Experimental valuesrepresent intensity values normalized to the value obtained from asingle dose of 0.25 mg/kg after 1 week.

FIG. 19 shows binding of ANTIBODY A COMPOSITION in relation to thenumber of successive biweekly doses at 0.1 mg/kg. After two applicationsmean intensity appeared increased, although the extent of immunostainingvaries considerably and therefore did not reach significance. After 4injections, amyloid-β plaques are immunostained more homogenously, butmean intensity is only slightly increased. Overall, the data forbiweekly application clearly indicate a tendency of increased plaquebinding that is correlated with the number of applications.

FIG. 20 shows binding of ANTIBODY A COMPOSITION in relation to thenumber of successive monthly doses at 0.15 mg/kg. Interestingly,comparable levels are obtained after 2 and 3 applications. This was notnecessarily expected and might indicate initiation of early effectswhich contribute to time-dependent differences in clearance mechanism,like delayed microglia cell activation.

The binding efficacy of ANTIBODY A COMPOSITION in relation toadministered dose is shown in FIGS. 21 and 22. Biweekly doses at 0.05,0.1 and 0.3 mg/kg (FIG. 21) and monthly doses of 0.075, 0.15 and 0.45mg/kg (FIG. 22) clearly showed a dose relationship. It is also evidentthat the response is not linear and additional factors like a temporallydelayed activation of microglia cells might contribute to the observednon-linearity.

It thus can be concluded that ANTIBODY A COMPOSITION-binding to mouse Aβplaques is dose-related with indications that multiple doses areaccumulative.

Example 17 Analysis of Antigen-Dependent Cellular Phagocytosis

In order to determine the ANTIBODY A COMPOSITION-mediated phagocyticeffect, genuine Aβ plaques from AD brain slices were pre-incubated withdifferent concentrations of ANTIBODY A COMPOSITION which comprisesmono-glycosylated ANTIBODY A and double-glycosylated ANTIBODY A and isdevoid of non-glycosylated ANTIBODY A as defined above and exposed toliving human primary monocytes.

Unfixed human AD brain tissue sections from the occipital cortex regionwere prepared from a severe Aβ case (Braak stage IV). Before addingliving cells sections were rehydrated with PBS for 5 minutes. ANTIBODY ACOMPOSITION antibodies were applied by incubation at definedconcentrations in PBS for 1 hour. After washing with PBS living cellswere added. Prestimulated human primary monocytes were used at 0.8 and1.5×106/ml in RPMI 1640 (Gibco # 61870-044) medium with 1% antibioticsolution from a stock solution containing 10,000 U/ml penicillin and10,000 mg/ml streptomycin (Gibco # 15140-122) and incubated at 37° C.with 5% carbon dioxide for 2 to 4 days. Methods for the preparation ofprestimulated human primary monocytes are well known in the art e.g. byuse of stimulating factors, like macrophage colony-stimulating factor(M-CSF).

After incubation, culture medium was gently removed and sectionspreserved by chemical fixation with 2% formaldehyde in PBS for 10minutes. Staining of residual Aβ plaque load was done by incubation withBAP-2, a mouse monoclonal antibody against Aβ conjugated to Alexa488fluorophore (Molecular Probes: A-20181, monoclonal antibody labelingkit) at a concentration of 10 mg/ml for 1 hour at room temperature.

Quantification of plaque removal was determined by measuring theimmunofluorescence of residual stained Aβ plaques. Images were recordedon a Leica TCS SP2 AOBS confocal laser scanning microscope. One opticallayer was recorded at excitation wavelength of 488 nm at a pinholesetting of 4 Airy using a HCX PL FL 20×/0.40 correction objective exceptin one experiment, where a HCPL Fluotar 10×/0.30 objective at a pinholesetting of 3 was used instead. Instrument settings were kept constantfor all images to allow a relational quantitative comparison.Specifically, laser power, gain and offset were adjusted to allow forsignal intensity monitoring within the dynamic range. For each ANTIBODYA COMPOSITION concentration grey matter regions were recorded atcomparable positions from consecutive sections in order to minimizefluctuations possibly coming from anatomical differences in plaque load.Potential competitive binding of ANTIBODY A COMPOSITION and thedetection antibody BAP-2, was measured in the absence of cells at allANTIBODY A COMPOSITION concentrations. An unrelated human IgG1 (Serotec,PHP010) antibody was used as an additional control. Images analysis wasperformed using the IMARIS Software (Bitplane, Switzerland). Theisosurface of BAP-2 positive pixels representing objects of BAP-2 sbound to the plaques were created by intensity thresholding. Surfacearea and total fluorescence intensity values were calculated using the“iso surface function” of the SurpassPro software module. Data wereexpressed as averaged staining area and total staining intensity valuesobtained from 5 grey matter regions of one brain section. The signalbaseline is composed by the instrumental noise and tissue scatter signalwas found negligible and was therefore not subtracted from the totalintensity signal.

The qualitative effect of ANTIBODY A COMPOSITION was visualized by adecrease in Aβ plaque stain that indicates increased phagocytosis of Aβplaques from human AD brain sections as shown in FIG. 23.Immunohistochemistry revealed a reduction in stainable Aβ plaquesclearly visible after a pre-incubation with 100 ng/ml ANTIBODY ACOMPOSITION already after 40 hours. The effect is very pronounced and atANTIBODY A COMPOSITION concentrations of 1 and 5 mg/ml. Aβ plaques aresubstantially and increasingly cleared by cellular phagocytosis withonly few large Aβ plaques remaining at 5 mg/ml. A quantitativemeasurement based on the immunoreactivity signals expressed as area andintensity of the same experiment is shown in FIG. 24.

Alternatively, the ANTIBODY A COMPOSITION-mediated phagocytic effect wasdetermined using Aβ conjugated fluorescent polystyrene beads. Therefore,fluorescent beads (3 mm, Fluoresbrite carboxy YG, Polysciences Inc.)were coupled with Aβ. Briefly, beads were washed 2× by suspension andcentrifugation in coupling buffer (50 mM MES buffer, pH 5.2, 1% DMSO).The pellet (approx. 10 ul) was suspended in 200 ul coupling buffer andactivated by addition of 20 ul of a 20% solution of EDC(Ethyl-diaminopropyl-carbodiimide, Pierce) in coupling buffer. Immediateaddition of 20 μg Aβ (1-40) or Aβ (1-42) (in 0.1% ammonium hydroxide,Bachem) started the coupling reaction. After one night incubation thebeads were washed with 3×0.5 ml 10 mM Tris.HCl pH 8.0 and 3×0.5 mlstorage buffer (10 mm Tris.HCl pH 8.0, 0.05% BSA, 0.05% NaN₃). The 1%suspension was stored at 4° C. until use. As a negative controlFluoresbrite carboxy NYO (red fluorescence) beads were coupled withall-D amino acid Aβ (1-40). (in 0.1% ammonium hydroxide, Bachem).

Murine monocytes/macrophages (cell line P388D1) were grown in C24transparent tissue culture clusters or C96 black microplates to approx.50% confluency. The culture medium was IMEM with 5% FBS, glutamine andantibiotics. To block unspecific scavenger receptors 10 ml fucoidan(Fluka, 10 mg/ml in water) was added to 200 ml culture volume andincubated for 2 hours. ANTIBODY A COMPOSITION was added in serialdilutions and pre-incubated for 30 minutes. Fluorescent Aβ beadsuspension (20 μl) was added and incubated for 3 hrs to allowphagocytosis. The adherent cells were washed vigorously 1× with ice coldEDTA and 2× with PBS in order to remove adhering agglutinates from thecell surface. The residual beads were either monitored by visualinspection at a Zeiss Axiovert 405 or for quantification by using amicroplate fluorimeter (Fluoroscan, Labsystems) with 444 nm (Exc) and485 nm (Em) filter settings

The qualitative effect of ANTIBODY A COMPOSITION on the phagocytosis ofsynthetic Aβ aggregates coupled to fluorescent fluorobeads by P388D1cells is shown in FIG. 25. The quantitative determination of the doseresponse of ANTIBODY A COMPOSITION is shown in FIG. 26. Two independentexperiments revealed a range from 30 to 200 ng/ml for EC50 and 10 to 60ng/ml for MEC. The observed variability is likely caused by differencesof the incubation stoichiometry, i.e. ratio of beads to cells. Theobserved decline of bead phagocytosis above a concentration >200 ng/mlis due to monovalent antibody interaction with limited antigen.

It thus can be concluded that ANTIBODY A COMPOSITION efficiently inducesphagocytosis of Aβ plaques from AD brain tissue sections in a doserelated manner.

1. A purified antibody molecule which comprises at least one antigen binding site comprising a glycosylated asparagine (Asn) in the variable region of a heavy chain (V_(H)).
 2. The purified antibody molecule of claim 1, wherein said glycosylated asparagine (Asn) in the variable region of the heavy chain (V_(H)) is in the CDR2 region.
 3. The purified antibody molecule of claim 1, wherein said glycosylated asparagine (Asn) in the variable region of the heavy chain (V_(H)) is in position 52 of SEQ ID NO: 2 or position 52 of SEQ ID NO:
 6. 4. The purified antibody molecule of claim 1, wherein said antibody molecule is capable of specifically recognizing the β-A4 peptide/Aβ4.
 5. The purified antibody molecule of claim 4, wherein said β-A4 peptide/Aβ4 has the following sequence: (SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

or a part of at least 15 amino acids of said sequence.
 6. The antibody molecule of claim 1, comprising a heavy chain (V_(H)) encoded by: (a) a nucleic acid molecule comprising the nucleotide sequence as shown in SEQ ID NO: 1 CAGGTGGAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGG CAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTA TGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGG TGAGCGCTATTAATGCTTCTGGTACTCGTACTTATTATGCTGATTCTGTT AAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCT GCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGC GCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTG ATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA;

(b) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2 (SEQ ID NO. 2) QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS;

(c) a nucleic acid molecule that hybridizes to the nucleic acid molecule of (a) or (b) and which encodes a polypeptide which is capable of binding to the β-A4 peptide/Aβ4 as shown in the following amino acid sequence (SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

 or is capable of binding to a fragment thereof which comprises at least amino acids; (d) a nucleic acid molecule that hybridizes to the nucleic acid molecule of (a) or (b) and which encodes a polypeptide which is capable of binding to at least two regions on the β-A4 peptide/Aβ4 as shown in the following amino acid sequence (SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

 or to at least two regions of a fragment of SEQ ID NO. 3 which comprises at least 15 amino acids wherein said two regions on the β-A4 peptide Aβ4 or said fragment thereof comprise the amino acids on position 3 to 6 and on position 18 to 26; or (e) a nucleic acid sequence that is degenerate to a nucleic acid sequence as defined in any one of (a) to (d).
 7. The antibody molecule of claim 1, wherein the variable region comprising a glycosylated asparagine (Asn) is comprised in a heavy chain selected from the group consisting of: (a) a heavy chain polypeptide encoded by a nucleic acid molecule as shown in SEQ ID NOS: 5, 23 or 25; (b) a heavy chain polypeptide having the amino acid sequence as shown in SEQ ID NO: 6 or 26; (c) a heavy chain polypeptide encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule defined in (a) and which encodes a polypeptide which is capable of binding to the β-A4 peptide/Aβ4 as shown in the following amino acid sequence (SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

 or a fragment thereof which comprises at least 15 amino acids; and (d) a heavy chain polypeptide encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule defined in (a) and which encodes a polypeptide which is capable of binding to at least two regions on the β-A4 peptide/Aβ4 as shown in the following amino acid sequence (SEQ ID NO: 3) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

 or to at least two regions of a fragment of SEQ ID NO. 3 which comprises at least 15 amino acids  wherein said two regions on the β-A4 peptide Aβ4 or said fragment thereof comprise the amino acids on position 3 to 6 and on position 18 to
 26. 8. The antibody molecule of claim 4, comprising two antigen binding sites, each antigen binding site comprising a glycosylated Asn in the respective variable region of the heavy chain (V_(H)).
 9. The antibody molecule of claim 8, wherein the CDR2 region of each of the heavy chains (V_(H)) comprises the glycosylated Asn.
 10. The antibody molecule of claim 9, wherein said Asn is Asn on position 52 of SEQ ID NO: 2 or is the Asn on position 52 of SEQ ID NO:
 6. 11. The antibody molecule of claim 1, wherein said glycosylation on Asn in the V_(H) region is selected from the group consisting of (a) a sugar structure of the biantennary complex type; (b) a sugar structure of the biantennary hybrid type; (c) a sugar structure of the biantennary oligomannose type; and (d) a sugar bi-antennary structure of the any of the structures as provided in FIG. 5 or FIG.
 27. 12. The antibody molecule of claim 11, wherein said sugar structure does not comprise a core fucosylation.
 13. The antibody molecule of claim 2, wherein said CDR-2 region has an amino acid sequence as shown in SEQ ID NO:
 12. 14. The antibody molecule of claim 4, which is recombinantly produced.
 15. The antibody molecule of claim 14 which is produced in a CHO-cell.
 16. The antibody molecule of claim 15, whereby wherein said CHO cell is CHO K1 or CHO K1 SV.
 17. A method for the preparation of an antibody molecule according to claim 1 comprising the steps of (a) recombinantly expressing a heterologous nucleic acid molecule encoding an antibody molecule as defined in claim 1 in a mammalian cultured cell; (b) purifying said recombinantly expressed antibody molecule by a method comprising the steps of (b1) protein A column purification; (b2) ion exchange column purification; and (b3) size exclusion column purification.
 18. The method of claim 17, wherein said ion exchange column purification comprises a cation exchange chromatography.
 19. The method of claim 17, further comprising as an additional step (c) an analytical chromatography and/or a further concentration step.
 20. A composition comprising a first antibody molecule wherein one antigen binding site of said first antibody molecule comprises a glycosylated asparagine (Asn) in the variable region of the heavy chain (V_(H)) and further comprising a second antibody molecule wherein the two antigen binding sites of said second antibody molecule comprise a glycosylated asparagine (Asn) in the variable region of the heavy chains (V_(H)), and wherein said composition comprises less than 5% of an antibody molecule wherein none of the antigen binding sites of said antibody molecule comprises a glycosylated asparagine (Asn) in the variable region of the heavy chain (V_(H)).
 21. The composition of claim 20 comprising an antibody molecule according to claim
 4. 22. The composition of claim 20, which is a pharmaceutical composition, and further comprises a pharmaceutically acceptable carrier or diluent.
 23. A method for the prevention and/or treatment of a disease associated with amyloidogenesis and/or amyloid-plaque formation comprising administering to a subject in need thereof an effective amount of an antibody molecule according to claim
 4. 24. A diagnostic kit for the detection of a disease associated with amyloidogenesis and/or amyloid-plaque formation comprising the antibody molecule according to claim
 4. 25. A method for the disintegration of β-amyloid plaques comprising administering to a subject in need thereof an effective amount of an antibody molecule according to claim
 4. 26. A method for passive immunization against β-amyloid plaque formation comprising administering to a subject in need thereof an effective amount of an antibody molecule according to claim
 4. 27. A method for the preventive treatment against a disease associated with amyloidogenesis and/or amyloid-plaque formation comprising administering to a subject in need thereof an effective amount of an antibody molecule according to claim
 4. 28. The method according to claim 27, wherein pre-existing plaques or aggregation intermediates of amyloid-β are to be reduced.
 29. A diagnostic kit for the diagnosis of a disease associated with amyloidogenesis and/or amyloid-plaque formation in a patient or for the diagnosis of the susceptibility of a patient for the development of a disease associated with amyloidogenesis and/or amyloid-plaque formation comprising the antibody molecule according to claim
 8. 30. The method according to claim 23, wherein said disease is dementia, Alzheimer's disease, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, hereditary cerebral hemorrhage with amyloidosis Dutch type, dementia associated with Lewy body formation, Parkinson's disease, HIV-related dementia, ALS or neuronal disorders related to aging.
 31. A kit comprising an antibody molecule of claim
 1. 32. A method for the prevention, treatment or amelioration of a disease associated with amyloidogenesis and/or amyloid-plaque formation, comprising administering an effective amount of the composition of claim 20 to a mammal in need thereof.
 33. The method of claim 32, wherein said disease is dementia, Alzheimer's disease, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, hereditary cerebral hemorrhage with amyloidosis Dutch type, dementia associated with Lewy body formation, Parkinson's disease, HIV-related dementia, ALS or neuronal disorders related to aging.
 34. The method of claim 32, wherein said mammal is a human.
 35. An antibody molecule comprising two light chains and two heavy chains, wherein each of the light chains comprises SEQ ID NO: 8 and each of the heavy chains comprises SEQ ID NO: 6, and wherein position 52 of SEQ ID NO: 6 in one of the heavy chains, but not both, is glycosylated.
 36. An antibody molecule comprising two light chains and two heavy chains, wherein each of the light chains comprises SEQ ID NO: 8 and each of the heavy chains comprises SEQ ID NO: 6, and wherein position 52 of SEQ ID NO: 6 in both of the heavy chains is glycosylated.
 37. A pharmaceutical composition comprising the antibody molecule of claim 35 and a pharmaceutically acceptable carrier or diluent.
 38. A pharmaceutical composition comprising the antibody molecule of claim 36 and a pharmaceutically acceptable carrier or diluent.
 39. A pharmaceutical composition comprising the antibody molecules of claims 35 and 36 and a pharmaceutically acceptable carrier or diluent.
 40. The pharmaceutical composition of claim 39, wherein less than 5% of the antibody molecules present in the composition are non-glycosylated in the variable region. 